Compositions and methods relating to a mutant clostridium difficile toxin

ABSTRACT

In one aspect, the invention relates to an immunogenic composition that includes a mutant  Clostridium difficile  toxin A and/or a mutant  Clostridium difficile  toxin B. The mutant toxin may include a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to the corresponding wild-type  C. difficile  toxin. The mutant toxins may include at least one amino acid that is chemically crosslinked. In another aspect, the invention relates to methods and compositions for use in culturing  Clostridium difficile  and in producing  C. difficile  toxins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase filing under 35 U.S.C. §371 ofInternational Application No. PCT/IB2013/059183 filed Oct. 7, 2013,which claims the benefit of U.S. Provisional Patent Application61/716,605, filed on Oct. 21, 2012. All of the foregoing applicationsare hereby incorporated by reference in their entireties.

FIELD

The present invention is directed to compositions and methods relatingto mutant Clostridium difficile toxins.

BACKGROUND

Clostridium difficile (C. difficile) is a Gram-positive anaerobicbacterium that is associated with gastrointestinal disease in humans.Colonization of C. difficile usually occurs in the colon if the naturalgut flora is diminished by treatment with antibiotics. An infection canlead to antibiotic-associated diarrhea and sometimes pseudomembranouscolitis through the secretion of the glucosylating toxins, toxin A andtoxin B (308 and 270 kDa, respectively), which are the primary virulencefactors of C. difficile.

Toxin A and toxin B are encoded within the 19 kb pathogenicity locus(PaLoc) by the genes tcdA and tcdB, respectively. Nonpathogenic strainsof C. difficile have this locus replaced by an alternative 115 base pairsequence.

Both toxin A and toxin B are potent cytotoxins. These proteins arehomologous glucosyltransferases that inactivate small GTPases of theRho/Rac/Ras family. The resulting disruption in signaling causes a lossof cell-cell junctions, dysregulation of the actin cytoskeleton, and/orapoptosis, resulting in the profound secretory diarrhea that isassociated with Clostridium difficile infections (CDI).

In the last decade, the numbers and severity of C. difficile outbreaksin hospitals, nursing homes, and other long-term care facilitiesincreased dramatically. Key factors in this escalation include emergenceof hypervirulent pathogenic strains, increased use of antibiotics,improved detection methods, and increased exposure to airborne spores inhealth care facilities.

Metronidazole and vancomycin represent the currently accepted standardof care for the antibiotic treatment of C. difficile associated disease(CDAD). However, about 20% of patients receiving such treatmentexperience a recurrence of infection after a first episode of CDI, andup to about 50% of those patients suffer from additional recurrences.Treatment of recurrences represents a very significant challenge, andthe majority of recurrences usually occur within one month of thepreceding episode.

Accordingly, there is a need for immunogenic and/or therapeuticcompositions and methods thereof directed to C. difficile.

SUMMARY OF THE INVENTION

These and other objectives are provided by the invention herein.

In one aspect, the invention relates to a mutant C. difficile toxin A.The mutant toxin A includes a mutation at residues positions 285, 287,700, 972, and 978 as compared to a wild-type toxin A. In one embodiment,the mutant toxin A includes SEQ ID NO: 183. In one embodiment, themutant toxin A is less cytotoxic than a corresponding wild-type toxin A.In one embodiment, the mutant toxin A includes at least one amino acidresidue that is chemically modified. In one aspect, the inventionrelates to an isolated polypeptide that includes SEQ ID NO: 183.

In another aspect, the invention relates to a mutant C. difficile toxinB. The mutant toxin B includes a mutation at residues 286, 288, 698,970, and 976 as compared to a wild-type toxin B. In one embodiment, themutant toxin B includes SEQ ID NO: 184. In one embodiment, the mutanttoxin B is less cytotoxic than a corresponding wild-type toxin B. In oneembodiment, the mutant toxin A includes at least one amino acid residuethat is chemically modified. In one aspect, the invention relates to anisolated polypeptide that includes SEQ ID NO: 184.

The invention further relates to compositions and methods for use inculturing C. difficile and producing C. difficile toxins. In one aspect,the invention relates to a culture medium including a vegetablehydrolysate and a C. difficile cell. In a preferred embodiment, thehydrolysate is soy hydrolysate. More preferably, the soy hydrolysate issoy hydrolysate SE50MK.

In another aspect, the invention relates to a culture medium including anitrogen source and a C. difficile cell. In one embodiment, the nitrogensource is a yeast extract. Preferably, the yeast extract is HY YEST 412(Kerry Biosciences).

In a further aspect, the invention relates to a culture medium includinga vegetable hydrolysate, yeast extract, and a C. difficile cell. In oneembodiment, the medium does not contain a carbon source.

In a preferred embodiment, the medium further includes a carbon source.The inventors discovered that fermentation of C. difficile in a culturemedium including at least one carbon source provided high OD₆₀₀ valuesand high toxin production yields, as compared to fermentation without acarbon source. In one embodiment, the carbon source is glucose,mannitol, fructose, and/or mannose.

In one embodiment, the C. difficile cell is not genetically modified. Inanother embodiment, the C. difficile cell is a recombinant C. difficilecell. In one embodiment, the C. difficile cell is lacks an endogenouspolynucleotide encoding a toxin. In another embodiment, the cellincludes a constitutive promoter. In a preferred embodiment, thepromoter is a Clostridium sporogenes feredoxin (fdx) promoter. In afurther embodiment, the cell does not include a native, regulatedchromosomal promoter.

In another aspect, the invention relates to a method of culturing C.difficile. The method includes culturing a C. difficile cell in amedium. In one embodiment, the medium includes soy hydrolysate and/oryeast extract. In a preferred embodiment, the medium further includes acarbon source. Preferably, the carbon source is glucose.

In one embodiment, the culturing step is carried out under anaerobicconditions.

In one embodiment, the C. difficile is grown as a seed culture. In oneembodiment, the seed culture is started by inoculation from a stockculture that was grown in the medium.

In one embodiment, the C. difficile is grown as a fermentation culture.In one embodiment, the fermentation culture was inoculated from a seedculture that was grown in the medium. In an alternative aspect, theinvention relates to a method of culturing C. difficile. The methodincludes culturing a C. difficile cell in a monoclonal antibody medium.

In one aspect, the invention relates to a method of producing a C.difficile toxin. The method includes culturing a C. difficile cell in amedium. The method further includes isolating a C. difficile toxin fromsaid medium.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A-H: Sequence alignment of wild-type C. difficile toxin A fromstrains 630, VPI10463, R20291, CD196, and mutant toxin A having SEQ IDNO: 4, using CLUSTALW alignment, default parameters.

FIG. 2 A-F: Sequence alignment of wild-type C. difficile toxin B fromstrains 630, VPI10463, R20291, CD196, and mutant toxin B having SEQ IDNO: 6, using CLUSTALW alignment, default parameters.

FIG. 3: Graph showing identification of wild-type toxin-negative C.difficile strains. Culture media of 13 C. difficile strains were testedby ELISA for toxin A. As illustrated, seven strains expressed toxin Aand 6 strains did not (strains 1351, 3232, 7322, 5036, 4811 and VPI11186).

FIGS. 4 A and B: SDS-PAGE results illustrating that triple mutant A (SEQID NO: 4), double mutant B (SEQ ID NO: 5), and triple mutant B (SEQ IDNO: 6) do not glucosylate Rac1 or RhoA GTPases in an in vitroglucosylation assays with UDP-¹⁴C-glucose; whereas 10 μg to 1 ng of wildtype toxin B does glucosylate Rac1.

FIG. 5: Western blot indicating abrogation of cysteine protease activityin mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively), ascompared to observation of cleaved fragments of wild-type toxins A and B(SEQ ID NOs: 1 and 2, respectively). See Example 13.

FIG. 6: Graphs showing that triple mutant toxins A and B (SEQ ID NOs: 4and 6, respectively) exhibit residual cytotoxicity when tested at highconcentrations (e.g., about 100 μg/ml) by in vitro cytotoxicity assay inIMR-90 cells.

FIG. 7: Graph showing that EC₅₀ values are similar for the triple mutanttoxin B (SEQ ID NO: 6) and hepta mutant toxin B (SEQ ID NO: 8).

FIG. 8: Graph representing results from in vitro cytotoxicity tests inwhich the ATP levels (RLUs) are plotted against increasingconcentrations of the triple mutant TcdA (SEQ ID NO: 4)(top panel) andtriple mutant TcdB (SEQ ID NO: 6)(bottom panel). Residual cytotoxicityof mutant toxin A and B can be completely abrogated with neutralizingantibodies specific for mutant toxin A (top panel-pAb A and mAbsA3-25+A60-22) and mutant toxin B (bottom panel-pAb B).

FIG. 9: Images of IMR-90 cell morphology at 72 hours post treatment.Panel A shows mock treated control cells. Panel B shows cell morphologyfollowing treatment with formalin inactivated mutant TcdB (SEQ ID NO:6). Panel C shows cell morphology following treatment with EDCinactivated mutant TcdB (SEQ ID NO: 6). Panel D shows cell morphologyfollowing treatment with wild-type toxin B (SEQ ID NO: 2). Panel E showscell morphology following treatment with triple mutant TcdB (SEQ ID NO:6). Similar results were observed for TcdA treatments.

FIG. 10: Graph showing neutralizing antibody titers as described inExample 25 (study muCdiff2010-06).

FIG. 11A-B: Graph showing neutralizing antibody titers as described inExample 26 (study muCdiff2010-07).

FIG. 12: Graph showing neutralizing antibody responses against toxins Aand B in hamsters after four immunizations as described in Example 27(study ham C. difficile 2010-02)

FIG. 13A-B: Graph showing neutralizing antibody responses in hamstersafter vaccination with chemically inactivated genetic mutant toxins andList Biological toxoids, as described in Example 27 (study ham C.difficile 2010-02).

FIG. 14: Survival curves for three immunized groups of hamsters ascompared to the non-immunized controls, described in Example 28 (studyham C. difficile 2010-02, continued).

FIG. 15: Graph showing relative neutralizing antibody response againstdifferent formulations of C. difficile mutant toxins in hamsters (studyham C. difficile 2010-03), as described in Example 29.

FIG. 16A-B: Graphs showing strong relative neutralizing antibodyresponse against chemically inactivated genetic mutant toxins A and B(SEQ ID NOs: 4 and 6, respectively) in cynomolgus macaques, as describedin Example 30.

FIG. 17: Amino acid sequences of variable regions of light (VL) andheavy (HL) chains of A3-25 mAb IgE. Signal peptide—highlighted;CDRs—italicized and underlined; Constant region—bolded and underlined(complete sequence not shown).

FIG. 18: Graph showing titration of individual toxin A monoclonalantibodies in the toxin neutralization assay using ATP levels(quantified by relative light units—RLU) as an indicator of cellviability. In comparison to the toxin (4×EC₅₀) control, mAbs A80-29,A65-33, A60-22 and A3-25 had increasing neutralizing effects on toxin Awith concentration but not to the level of the positive rabbitanti-toxin A control, mAbs A50-10, A56-33, and A58-46 did not neutralizetoxin A. The cell only control was 1-1.5×10⁶ RLUs.

FIG. 19: Mapping of 8 epitope groups of toxin B mAbs by BiaCore

FIG. 20A-C: Synergistic neutralizing activities of combinations of toxinA mAbs: Adding different dilutions of neutralizing antibodies A60-22,A65-33, and A80-29 to increasing concentrations of A3-25 mAbsynergistically increased the neutralization of toxin A regardless ofthe dilution. The RLUs of the toxin A only (4×EC₅₀) control isillustrated (<0.3×10⁶) and cell only controls were 2-2.5×10⁶ RLUs asdepicted in graphs shown in FIG. 20B and FIG. 20C.

FIG. 21: Synergistic neutralizing activities of toxin B mAbs:Neutralization of toxin B by mAbs 8-26, B60-2 and B59-3 is illustratedin FIG. 21A. Neutralization of toxin B is synergistically increasedafter combining B8-26 with dilutions of B59-3 (FIG. 21B)

FIG. 22: Western blot showing that Rac1 GTPase expression is reduced ingenetic mutant toxin B (SEQ ID NO: 6) extracts from 24 to 96 hours, butnot in wild-type toxin B (SEQ ID NO: 2) treated extracts. The blot alsoshows that Rac1 is glucosylated in toxin B-treated extracts, but not ingenetic mutant toxin B treated extracts.

FIG. 23A-K: Graph representing results from in vitro cytotoxicity testsin which the ATP levels (RLUs) are plotted against increasingconcentrations of C. difficile culture media and the hamster serum pool(┘); crude toxin (culture harvest) from the respective strain and thehamster serum pool (); purified toxin (commercial toxin obtained fromList Biologicals) and the hamster serum pool (▴); crude toxin (▾),control; and purified toxin (♦), control. The toxins from the respectivestrains were added to the cells at 4×EC₅₀ values. FIG. 23 shows that animmunogenic composition including mutant TcdA (SEQ ID NO: 4) and mutantTcdB (SEQ ID NO: 6), wherein the mutant toxins were inactivated withEDC, according to, for example, Example 29, Table 15, described herein,induced neutralizing antibodies that exhibited neutralizing activityagainst toxins from at least the following 16 different CDC strains ofC. difficile, in comparison to the respective toxin only control:2007886 (FIG. 23A); 2006017 (FIG. 23B); 2007070 (FIG. 23C); 2007302(FIG. 23D); 2007838 (FIG. 23E); 2007886 (FIG. 23F); 2009292 (FIG. 23G);2004013 (FIG. 23H); 2009141 (FIG. 23I); 2005022 (FIG. 23J); 2006376(FIG. 23K).

FIG. 24: Illustration of an exemplary EDC/NHS inactivation of mutant C.difficile toxins, resulting in at least three possible types ofmodifications: crosslinks, glycine adducts, and beta-alanine adducts.Panel A illustrates crosslinking. Carboxylic residues of triple mutanttoxins are activated by the addition of EDC and NHS. The activatedesters react with primary amines to form stable amide bonds, resultingin intra- and intermolecular crosslinks. Panel B illustrates formationof glycine adducts. After inactivation, residual activated esters arequenched by the addition of glycine to form stable amide bonds. Panel Cillustrates formation of beta-alanine adducts. Three moles of NHS canreact with one mole of EDC to form activated beta-alanine. This thenreacts with primary amines to form stable amide bonds.

FIG. 25: Illustration of an exemplary EDC/NHS inactivation of mutant C.difficile toxins, resulting in at least one of the following types ofmodifications: (A) crosslinks, (B) glycine adducts, and (C) beta-alanineadducts.

FIG. 26: Graph representing results from an in vitro cytotoxicity assayin which the ATP levels (RLUs) (72 hr ATP) are plotted againstincreasing concentrations of the wild-type TcdB, commercially obtainedfrom List Biologicals (□), triple mutant TcdB(SEQ ID NO: 86)(), andpenta mutant TcdB (SEQ ID NO: 184) (▪). IMR-90 cells (

) were used as control.

FIG. 27: Graph showing competitive inhibition of triple mutant toxin B(SEQ ID NO: 86)-mediated cytotoxicity by penta mutant toxin B (SEQ IDNO: 184) on IMR −90 cells, (72 hr ATP assay). -- represents pentamutant toxin B (SEQ ID NO: 184)(“PM-B”); -▴- represents triple mutant(TM) at 200 ng/mL.

FIG. 28: Graph showing final OD and triple mutant toxin B titer (mg/I)following a perfusion fermentation (CDF-5126). -- representsOD_(600 nm); -▴-represents perfusion flow rate (Fermentor volumes/2.0h); -▪- represents glucose (g/L); --represents toxoid B (triple mutant,SEQ ID NO: 86)

FIG. 29: Graph showing final OD and triple mutant toxin B titer (mg/I)results from another perfusion culture (CDF-5127). -- representsOD_(600 nm); -▴-represents perfusion flow rate (Fermentor volumes/2.0h); -▪- represents glucose (g/L); --represents toxoid B (triple mutant,SEQ ID NO: 86).

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 sets forth the amino acid sequence for wild-type C.difficile 630 toxin A (TcdA).

SEQ ID NO: 2 sets forth the amino acid sequence for wild-type C.difficile 630 toxin B (TcdB).

SEQ ID NO: 3 sets forth the amino acid sequence for a mutant TcdA havinga mutation at positions 285 and 287, as compared to SEQ ID NO: 1.

SEQ ID NO: 4 sets forth the amino acid sequence for a mutant TcdA havinga mutation at positions 285, 287, and 700, as compared to SEQ ID NO: 1.

SEQ ID NO: 5 sets forth the amino acid sequence for a mutant TcdB havinga mutation at positions 286 and 288, as compared to SEQ ID NO: 2.

SEQ ID NO: 6 sets forth the amino acid sequence for a mutant TcdB havinga mutation at positions 286, 288, and 698, as compared to SEQ ID NO: 2.

SEQ ID NO: 7 sets forth the amino acid sequence for a mutant TcdA havinga mutation at positions 269, 272, 285, 287, 460, 462, and 700, ascompared to SEQ ID NO: 1

SEQ ID NO: 8 sets forth the amino acid sequence for a mutant TcdB havinga mutation at positions 270, 273, 286, 288, 461, 463, and 698, ascompared to SEQ ID NO: 2

SEQ ID NO: 9 sets forth a DNA sequence encoding a wild-type C. difficile630 toxin A (TcdA).

SEQ ID NO: 10 sets forth a DNA sequence encoding a wild-type C.difficile 630 toxin B (Tcd B).

SEQ ID NO: 11 sets forth a DNA sequence encoding SEQ ID NO: 3

SEQ ID NO: 12 sets forth a DNA sequence encoding SEQ ID NO: 4

SEQ ID NO: 13 sets forth a DNA sequence encoding SEQ ID NO: 5

SEQ ID NO: 14 sets forth a DNA sequence encoding SEQ ID NO: 6

SEQ ID NO: 15 sets forth the amino acid sequence for wild-type C.difficile R20291 TcdA.

SEQ ID NO: 16 sets forth a DNA sequence encoding SEQ ID NO: 15.

SEQ ID NO: 17 sets forth the amino acid sequence for wild-type C.difficile CD196 TcdA.

SEQ ID NO: 18 sets forth a DNA sequence encoding SEQ ID NO: 17.

SEQ ID NO: 19 sets forth the amino acid sequence for wild-type C.difficile VPI10463 TcdA.

SEQ ID NO: 20 sets forth a DNA sequence encoding SEQ ID NO: 19.

SEQ ID NO: 21 sets forth the amino acid sequence for wild-type C.difficile R20291 TcdB.

SEQ ID NO: 22 sets forth a DNA sequence encoding SEQ ID NO: 21.

SEQ ID NO: 23 sets forth the amino acid sequence for wild-type C.difficile CDI 96 TcdB.

SEQ ID NO: 24 sets forth a DNA sequence encoding SEQ ID NO: 23.

SEQ ID NO: 25 sets forth the amino acid sequence for wild-type C.difficile VPI10463 TcdB.

SEQ ID NO: 26 sets forth a DNA sequence encoding SEQ ID NO: 25.

SEQ ID NO: 27 sets forth a DNA sequence of a pathogenicity locus ofwild-type C. difficile VPI10463.

SEQ ID NO: 28 sets forth the amino acid sequence for residues 101 to 293of SEQ ID NO: 1.

SEQ ID NO: 29 sets forth the amino acid sequence for residues 1 to 542of SEQ ID NO: 1.

SEQ ID NO: 30 sets forth the amino acid sequence for residues 101 to 293of SEQ ID NO: 2.

SEQ ID NO: 31 sets forth the amino acid sequence for residues 1 to 543of SEQ ID NO: 2.

SEQ ID NO: 32 sets forth the amino acid sequence for residues 543 to 809of SEQ ID NO: 1.

SEQ ID NO: 33 sets forth the amino acid sequence for residues 544 to 767of SEQ ID NO: 2.

SEQ ID NO: 34 sets forth the amino acid sequence for a mutant TcdA,wherein residues 101, 269, 272, 285, 287, 460, 462, 541, 542, 543, 589,655, and 700 may be any amino acid.

SEQ ID NO: 35 sets forth the amino acid sequence for a mutant TcdB,wherein 102, 270, 273, 286, 288, 384, 461, 463, 520, 543, 544, 587, 600,653, 698, and 751 may be any amino acid.

SEQ ID NO: 36 sets forth the amino acid sequence for the variable lightchain of a neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 37 sets forth the amino acid sequence for the variable heavychain of a neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 38 sets forth the amino acid sequence for CDR1 of thevariable light chain of neutralizing antibody of C. difficile TcdA(A3-25 mAb).

SEQ ID NO: 39 sets forth the amino acid sequence for CDR2 of thevariable light chain of neutralizing antibody of C. difficile TcdA(A3-25 mAb).

SEQ ID NO: 40 sets forth the amino acid sequence for CDR3 of thevariable light chain of neutralizing antibody of C. difficile TcdA(A3-25 mAb).

SEQ ID NO: 41 sets forth the amino acid sequence for CDR1 of thevariable heavy chain of neutralizing antibody of C. difficile TcdA(A3-25 mAb).

SEQ ID NO: 42 sets forth the amino acid sequence for CDR2 of thevariable heavy chain of neutralizing antibody of C. difficile TcdA(A3-25 mAb).

SEQ ID NO: 43 sets forth the amino acid sequence for CDR3 of thevariable heavy chain of neutralizing antibody of C. difficile TcdA(A3-25 mAb).

SEQ ID NO: 44 sets forth a DNA sequence encoding SEQ ID NO: 3.

SEQ ID NO: 45 sets forth a DNA sequence encoding SEQ ID NO: 4.

SEQ ID NO: 46 sets forth a DNA sequence encoding SEQ ID NO: 5.

SEQ ID NO: 47 sets forth a DNA sequence encoding SEQ ID NO: 6.

SEQ ID NO: 48 sets forth the nucleotide sequence of immunostimulatoryoligonucleotide ODN CpG 24555.

SEQ ID NO: 49 sets forth the amino acid sequence for the variable heavychain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 50 sets forth the amino acid sequence for the signal peptideof the variable heavy chain of a C. difficile TcdB neutralizing antibody(B8-26 mAb).

SEQ ID NO: 51 sets forth the amino acid sequence for CDR1 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 52 sets forth the amino acid sequence for CDR2 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 53 sets forth the amino acid sequence for CDR3 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 54 sets forth the amino acid sequence for the constant regionof the variable heavy chain of a C. difficile TcdB neutralizing antibody(B8-26 mAb).

SEQ ID NO: 55 sets forth the amino acid sequence for the variable lightchain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 56 sets forth the amino acid sequence for the signal peptideof the variable light chain of a C. difficile TcdB neutralizing antibody(B8-26 mAb).

SEQ ID NO: 57 sets forth the amino acid sequence for CDR1 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 58 sets forth the amino acid sequence for CDR2 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 59 sets forth the amino acid sequence for CDR3 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 60 sets forth the amino acid sequence for the variable heavychain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 61 sets forth the amino acid sequence for the signal peptideof the variable heavy chain of a C. difficile TcdB neutralizing antibody(B59-3 mAb).

SEQ ID NO: 62 sets forth the amino acid sequence for CDR1 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 63 sets forth the amino acid sequence for CDR2 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 64 sets forth the amino acid sequence for CDR3 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 65 sets forth the amino acid sequence for the constant regionof the variable heavy chain of a C. difficile TcdB neutralizing antibody(B59-3 mAb).

SEQ ID NO: 66 sets forth the amino acid sequence for the variable lightchain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 67 sets forth the amino acid sequence for the signal peptideof the variable light chain of a C. difficile TcdB neutralizing antibody(B59-3 mAb).

SEQ ID NO: 68 sets forth the amino acid sequence for CDR1 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 69 sets forth the amino acid sequence for CDR2 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 70 sets forth the amino acid sequence for CDR3 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 71 sets forth the amino acid sequence for the variable heavychain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 72 sets forth the amino acid sequence for the signal peptideof the variable heavy chain of a C. difficile TcdB neutralizing antibody(B9-30 mAb).

SEQ ID NO: 73 sets forth the amino acid sequence for CDR1 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 74 sets forth the amino acid sequence for CDR2 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 75 sets forth the amino acid sequence for CDR3 of thevariable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 76 sets forth the amino acid sequence for the constant regionof the variable heavy chain of a C. difficile TcdB neutralizing antibody(B9-30 mAb).

SEQ ID NO: 77 sets forth the amino acid sequence for the variable lightchain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 78 sets forth the amino acid sequence for the signal peptideof the variable light chain of a C. difficile TcdB neutralizing antibody(B9-30 mAb).

SEQ ID NO: 79 sets forth the amino acid sequence for CDR1 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 80 sets forth the amino acid sequence for CDR2 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 81 sets forth the amino acid sequence for CDR3 of thevariable light chain of a C. difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 82 sets forth the amino acid sequence for a mutant TcdB,wherein a residue at positions 102, 270, 273, 286, 288, 384, 461, 463,520, 543, 544, 587, 600, 653, 698, and 751 may be any amino acid.

SEQ ID NO: 83 sets forth the amino acid sequence for a mutant TcdAhaving a mutation at positions 269, 272, 285, 287, 460, 462, and 700, ascompared to SEQ ID NO: 1, wherein the methionine at position 1 isabsent.

SEQ ID NO: 84 sets forth the amino acid sequence for a mutant C.difficile toxin A having a mutation at positions 285, 287, and 700, ascompared to SEQ ID NO: 1, wherein the methionine at position 1 isabsent.

SEQ ID NO: 85 sets forth the amino acid sequence for a mutant C.difficile toxin B having a mutation at positions 270, 273, 286, 288,461, 463, and 698, as compared to SEQ ID NO: 2, wherein the methionineat position 1 is absent.

SEQ ID NO: 86 sets forth the amino acid sequence for a mutant C.difficile toxin B having a mutation at positions 286, 288, and 698, ascompared to SEQ ID NO: 2, wherein the methionine at position 1 isabsent.

SEQ ID NO: 87 sets forth the amino acid sequence for wild-type C.difficile 2004013 TcdA.

SEQ ID NO: 88 sets forth the amino acid sequence for wild-type C.difficile 2004111 TcdA.

SEQ ID NO: 89 sets forth the amino acid sequence for wild-type C.difficile 2004118 TcdA.

SEQ ID NO: 90 sets forth the amino acid sequence for wild-type C.difficile 2004205 TcdA.

SEQ ID NO: 91 sets forth the amino acid sequence for wild-type C.difficile 2004206 TcdA.

SEQ ID NO: 92 sets forth the amino acid sequence for wild-type C.difficile 2005022 TcdA.

SEQ ID NO: 93 sets forth the amino acid sequence for wild-type C.difficile 2005088 TcdA.

SEQ ID NO: 94 sets forth the amino acid sequence for wild-type C.difficile 2005283 TcdA.

SEQ ID NO: 95 sets forth the amino acid sequence for wild-type C.difficile 2005325 TcdA.

SEQ ID NO: 96 sets forth the amino acid sequence for wild-type C.difficile 2005359 TcdA.

SEQ ID NO: 97 sets forth the amino acid sequence for wild-type C.difficile 2006017 TcdA.

SEQ ID NO: 98 sets forth the amino acid sequence for wild-type C.difficile 2007070 TcdA.

SEQ ID NO: 99 sets forth the amino acid sequence for wild-type C.difficile 2007217 TcdA.

SEQ ID NO: 100 sets forth the amino acid sequence for wild-type C.difficile 2007302 TcdA.

SEQ ID NO: 101 sets forth the amino acid sequence for wild-type C.difficile 2007816 TcdA.

SEQ ID NO: 102 sets forth the amino acid sequence for wild-type C.difficile 2007838 TcdA.

SEQ ID NO: 103 sets forth the amino acid sequence for wild-type C.difficile 2007858 TcdA.

SEQ ID NO: 104 sets forth the amino acid sequence for wild-type C.difficile 2007886 TcdA.

SEQ ID NO: 105 sets forth the amino acid sequence for wild-type C.difficile 2008222 TcdA.

SEQ ID NO: 106 sets forth the amino acid sequence for wild-type C.difficile 2009078 TcdA.

SEQ ID NO: 107 sets forth the amino acid sequence for wild-type C.difficile 2009087 TcdA.

SEQ ID NO: 108 sets forth the amino acid sequence for wild-type C.difficile 2009141 TcdA.

SEQ ID NO: 109 sets forth the amino acid sequence for wild-type C.difficile 2009292 TcdA.

SEQ ID NO: 110 sets forth the amino acid sequence for wild-type C.difficile 2004013 TcdB.

SEQ ID NO: 111 sets forth the amino acid sequence for wild-type C.difficile 2004111 TcdB.

SEQ ID NO: 112 sets forth the amino acid sequence for wild-type C.difficile 2004118 TcdB.

SEQ ID NO: 113 sets forth the amino acid sequence for wild-type C.difficile 2004205 TcdB.

SEQ ID NO: 114 sets forth the amino acid sequence for wild-type C.difficile 2004206 TcdB.

SEQ ID NO: 115 sets forth the amino acid sequence for wild-type C.difficile 2005022 TcdB.

SEQ ID NO: 116 sets forth the amino acid sequence for wild-type C.difficile 2005088 TcdB.

SEQ ID NO: 117 sets forth the amino acid sequence for wild-type C.difficile 2005283 TcdB.

SEQ ID NO: 118 sets forth the amino acid sequence for wild-type C.difficile 2005325 TcdB.

SEQ ID NO: 119 sets forth the amino acid sequence for wild-type C.difficile 2005359 TcdB.

SEQ ID NO: 120 sets forth the amino acid sequence for wild-type C.difficile 2006017 TcdB.

SEQ ID NO: 121 sets forth the amino acid sequence for wild-type C.difficile 2006376 TcdB.

SEQ ID NO: 122 sets forth the amino acid sequence for wild-type C.difficile 2007070 TcdB.

SEQ ID NO: 123 sets forth the amino acid sequence for wild-type C.difficile 2007217 TcdB.

SEQ ID NO: 124 sets forth the amino acid sequence for wild-type C.difficile 2007302 TcdB.

SEQ ID NO: 125 sets forth the amino acid sequence for wild-type C.difficile 2007816 TcdB.

SEQ ID NO: 126 sets forth the amino acid sequence for wild-type C.difficile 2007838 TcdB.

SEQ ID NO: 127 sets forth the amino acid sequence for wild-type C.difficile 2007858 TcdB.

SEQ ID NO: 128 sets forth the amino acid sequence for wild-type C.difficile 2007886 TcdB.

SEQ ID NO: 129 sets forth the amino acid sequence for wild-type C.difficile 2008222 TcdB.

SEQ ID NO: 130 sets forth the amino acid sequence for wild-type C.difficile 2009078 TcdB.

SEQ ID NO: 131 sets forth the amino acid sequence for wild-type C.difficile 2009087 TcdB.

SEQ ID NO: 132 sets forth the amino acid sequence for wild-type C.difficile 2009141 TcdB.

SEQ ID NO: 133 sets forth the amino acid sequence for wild-type C.difficile 2009292 TcdB.

SEQ ID NO: 134 sets forth the amino acid sequence for wild-type C.difficile 014 TcdA.

SEQ ID NO: 135 sets forth the amino acid sequence for wild-type C.difficile 015 TcdA.

SEQ ID NO: 136 sets forth the amino acid sequence for wild-type C.difficile 020 TcdA.

SEQ ID NO: 137 sets forth the amino acid sequence for wild-type C.difficile 023 TcdA.

SEQ ID NO: 138 sets forth the amino acid sequence for wild-type C.difficile 027 TcdA.

SEQ ID NO: 139 sets forth the amino acid sequence for wild-type C.difficile 029 TcdA.

SEQ ID NO: 140 sets forth the amino acid sequence for wild-type C.difficile 046 TcdA.

SEQ ID NO: 141 sets forth the amino acid sequence for wild-type C.difficile 014 TcdB.

SEQ ID NO: 142 sets forth the amino acid sequence for wild-type C.difficile 015 TcdB.

SEQ ID NO: 143 sets forth the amino acid sequence for wild-type C.difficile 020 TcdB.

SEQ ID NO: 144 sets forth the amino acid sequence for wild-type C.difficile 023 TcdB.

SEQ ID NO: 145 sets forth the amino acid sequence for wild-type C.difficile 027 TcdB.

SEQ ID NO: 146 sets forth the amino acid sequence for wild-type C.difficile 029 TcdB.

SEQ ID NO: 147 sets forth the amino acid sequence for wild-type C.difficile 046 TcdB.

SEQ ID NO: 148 sets forth the amino acid sequence for wild-type C.difficile 001 TcdA.

SEQ ID NO: 149 sets forth the amino acid sequence for wild-type C.difficile 002 TcdA.

SEQ ID NO: 150 sets forth the amino acid sequence for wild-type C.difficile 003 TcdA.

SEQ ID NO: 151 sets forth the amino acid sequence for wild-type C.difficile 004 TcdA.

SEQ ID NO: 152 sets forth the amino acid sequence for wild-type C.difficile 070 TcdA.

SEQ ID NO: 153 sets forth the amino acid sequence for wild-type C.difficile 075 TcdA.

SEQ ID NO: 154 sets forth the amino acid sequence for wild-type C.difficile 077 TcdA.

SEQ ID NO: 155 sets forth the amino acid sequence for wild-type C.difficile 081 TcdA.

SEQ ID NO: 156 sets forth the amino acid sequence for wild-type C.difficile 117 TcdA.

SEQ ID NO: 157 sets forth the amino acid sequence for wild-type C.difficile 131 TcdA.

SEQ ID NO: 158 sets forth the amino acid sequence for wild-type C.difficile 001 TcdB.

SEQ ID NO: 159 sets forth the amino acid sequence for wild-type C.difficile 002 TcdB.

SEQ ID NO: 160 sets forth the amino acid sequence for wild-type C.difficile 003 TcdB.

SEQ ID NO: 161 sets forth the amino acid sequence for wild-type C.difficile 004 TcdB.

SEQ ID NO: 162 sets forth the amino acid sequence for wild-type C.difficile 070 TcdB.

SEQ ID NO: 163 sets forth the amino acid sequence for wild-type C.difficile 075 TcdB.

SEQ ID NO: 164 sets forth the amino acid sequence for wild-type C.difficile 077 TcdB.

SEQ ID NO: 165 sets forth the amino acid sequence for wild-type C.difficile 081 TcdB.

SEQ ID NO: 166 sets forth the amino acid sequence for wild-type C.difficile 117 TcdB.

SEQ ID NO: 167 sets forth the amino acid sequence for wild-type C.difficile 131 TcdB.

SEQ ID NO: 168 sets forth the amino acid sequence for wild-type C.difficile 053 TcdA.

SEQ ID NO: 169 sets forth the amino acid sequence for wild-type C.difficile 078 TcdA.

SEQ ID NO: 170 sets forth the amino acid sequence for wild-type C.difficile 087 TcdA.

SEQ ID NO: 171 sets forth the amino acid sequence for wild-type C.difficile 095 TcdA.

SEQ ID NO: 172 sets forth the amino acid sequence for wild-type C.difficile 126 TcdA.

SEQ ID NO: 173 sets forth the amino acid sequence for wild-type C.difficile 053 TcdB.

SEQ ID NO: 174 sets forth the amino acid sequence for wild-type C.difficile 078 TcdB.

SEQ ID NO: 175 sets forth the amino acid sequence for wild-type C.difficile 087 TcdB.

SEQ ID NO: 176 sets forth the amino acid sequence for wild-type C.difficile 095 TcdB.

SEQ ID NO: 177 sets forth the amino acid sequence for wild-type C.difficile 126 TcdB.

SEQ ID NO: 178 sets forth the amino acid sequence for wild-type C.difficile 059 TcdA.

SEQ ID NO: 179 sets forth the amino acid sequence for wild-type C.difficile 059 TcdB.

SEQ ID NO: 180 sets forth the amino acid sequence for wild-type C.difficile 106 TcdA.

SEQ ID NO: 181 sets forth the amino acid sequence for wild-type C.difficile 106 TcdB.

SEQ ID NO: 182 sets forth the amino acid sequence for wild-type C.difficile 017 TcdB.

SEQ ID NO: 183 sets forth the amino acid sequence for a mutant TcdAhaving a mutation at positions 285, 287, 700, 972, and 978 as comparedto SEQ ID NO: 1.

SEQ ID NO: 184 sets forth the amino acid sequence for a mutant TcdBhaving a mutation at positions 286, 288, 698, 970, and 976 as comparedto SEQ ID NO: 2.

SEQ ID NO: 185 through SEQ ID NO: 195 each set forth the amino acidsequence for an exemplary mutant toxin.

SEQ ID NO: 196 through SEQ ID NO: 212 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 213 through SEQ ID NO: 222 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 223 through SEQ ID NO: 236 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 237 through SEQ ID NO: 243 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 244 through SEQ ID NO: 245 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 246 through SEQ ID NO: 249 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 250 through SEQ ID NO: 253 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 254 sets forth the amino acid sequence for an exemplarymutant toxin.

SEQ ID NO: 255 through SEQ ID NO: 263 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 264 through SEQ ID NO: 269 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 270 through SEQ ID NO: 275 each set forth the amino acidsequence for an exemplary mutant toxin.

SEQ ID NO: 276 through SEQ ID NO: 323 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 324 through SEQ ID NO: 373 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 374 through SEQ ID NO: 421 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 422 through SEQ ID NO: 471 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 472 through SEQ ID NO: 519 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 568 through SEQ ID NO: 615 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 520 through SEQ ID NO: 567 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 616 through SEQ ID NO: 663 each set forth the amino acidsequence for an exemplary mutant toxin B.

SEQ ID NO: 664 through SEQ ID NO: 711 each set forth the amino acidsequence for an exemplary mutant toxin A.

SEQ ID NO: 712 through SEQ ID NO: 761 each set forth the amino acidsequence for an exemplary mutant toxin B.

DETAILED DESCRIPTION

The inventors surprisingly discovered, among other things, a mutant C.difficile toxin A and toxin B, and methods thereof. The mutants arecharacterized, in part, by being immunogenic and exhibiting reducedcytotoxicity compared to a wild-type form of the respective toxin. Thepresent invention also relates to immunogenic portions thereof,biological equivalents thereof, and isolated polynucleotides thatinclude nucleic acid sequences encoding any of the foregoing.

The immunogenic compositions described herein unexpectedly demonstratedthe ability to elicit novel neutralizing antibodies against C. difficiletoxins and they may have the ability to confer active and/or passiveprotection against a C. difficile challenge. The novel antibodies aredirected against various epitopes of toxin A and toxin B. The inventorsfurther discovered that a combination of at least two of theneutralizing monoclonal antibodies can exhibit an unexpectedlysynergistic effect in respective in vitro neutralization of toxin A andtoxin B.

The inventive compositions described herein may be used to treat,prevent, decrease the risk of, decrease occurrences of, decreaseseverity of, and/or delay the outset of a C. difficile infection, C.difficile associated disease (CDAD), syndrome, condition, symptom,and/or complication thereof in a mammal, as compared to a mammal towhich the composition was not administered.

Moreover, the inventors discovered a recombinant asporogenic C.difficile cell that can stably express the mutant C. difficile toxin Aand toxin B, and novel methods for producing the same.

Immunogenic Compositions

In one aspect, the invention relates to an immunogenic composition thatincludes a mutant C. difficile toxin. The mutant C. difficile toxinincludes an amino acid sequence having at least one mutation in aglucosyltransferase domain and at least one mutation in a cysteineprotease domain, relative to the corresponding wild-type C. difficiletoxin.

The term “wild-type,” as used herein, refers to the form found innature. For example, a wild-type polypeptide or polynucleotide sequenceis a sequence present in an organism that can be isolated from a sourcein nature and which has not been intentionally modified by humanmanipulation. The present invention also relates to isolatedpolynucleotides that include nucleic acid sequences encoding any of theforegoing. In addition, the present invention relates to use of any ofthe foregoing compositions to treat, prevent, decrease the risk of,decrease severity of, decrease occurrences of, and/or delay the outsetof a C. difficile infection, C. difficile associated disease, syndrome,condition, symptom, and/or complication thereof in a mammal, as comparedto a mammal to which the composition is not administered, as well asmethods for preparing said compositions.

As used herein, an “immunogenic composition” or “immunogen” refers to acomposition that elicits an immune response in a mammal to which thecomposition is administered.

An “immune response” refers to the development of a beneficial humoral(antibody mediated) and/or a cellular (mediated by antigen-specific Tcells or their secretion products) response directed against a C.difficile toxin in a recipient patient. The immune response may behumoral, cellular, or both.

The immune response can be an active response induced by administrationof an immunogenic composition, an immunogen. Alternatively, the immuneresponse can be a passive response induced by administration of antibodyor primed T-cells.

The presence of a humoral (antibody-mediated) immune response can bedetermined, for example, by cell-based assays known in the art, such asa neutralizing antibody assay, ELISA, etc.

A cellular immune response is typically elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4+T helper cells and/orCD8+cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4+T cells) or CTL (cytotoxic Tlymphocyte) assays known in the art.

In one embodiment, an immunogenic composition is a vaccine composition.As used herein, a “vaccine composition” is a composition that elicits animmune response in a mammal to which the composition is administered.The vaccine composition may protect the immunized mammal againstsubsequent challenge by an immunizing agent or an immunologicallycross-reactive agent. Protection can be complete or partial with regardto reduction in symptoms or infection as compared to a non-vaccinatedmammal under the same conditions.

The immunogenic compositions described herein are cross-reactive, whichrefers to having a characteristic of being able to elicit an effectiveimmune response (e.g., humoral immune response) against a toxin producedby another C. difficile strain that is different from the strain fromwhich the composition is derived. For example, the immunogeniccompositions (e.g., derived from C. difficile 630) described herein mayelicit cross-reactive antibodies that can bind to toxins produced bymultiple strains of C. difficile (e.g., toxins produced by C. difficileR20291 and VPI10463). See, for example, Example 37. Cross-reactivity isindicative of the cross-protection potential of the bacterial immunogen,and vice versa.

The term “cross-protective” as used herein refers to the ability of theimmune response induced by an immunogenic composition to prevent orattenuate infection by a different bacterial strain or species of thesame genus. For example, an immunogenic composition (e.g., derived fromC. difficile 630) described herein may induce an effective immuneresponse in a mammal to attenuate a C. difficile infection and/or toattenuate a C. difficile disease caused by a strain other than 630(e.g., C. difficile R20291) in the mammal.

Exemplary mammals in which the immunogenic composition or immunogenelicits an immune response include any mammals, such as, for example,mice, hamsters, primates, and humans. In a preferred embodiment, theimmunogenic composition or immunogen elicits an immune response in ahuman to which the composition is administered.

As described above, toxin A (TcdA) and toxin B (TcdB) are homologousglucosyltransferases that inactivate small GTPases of the Rho/Rac/Rasfamily. The action of TcdA and TcdB on mammalian target cells depends ona multistep mechanism of receptor-mediated endocytosis, membranetranslocation, autoproteolytic processing, and monoglucosylation ofGTPases. Many of these functional activities have been ascribed todiscrete regions within the primary sequence of the toxins, and thetoxins have been imaged to show that these molecules are similar instructure.

The wild-type gene for TcdA has about 8130 nucleotides that encode aprotein having a deduced molecular weight of about 308-kDa, having about2710 amino acids. As used herein, a wild-type C. difficile TcdA includesa C. difficile TcdA from any wild-type C. difficile strain. A wild-typeC. difficile TcdA may include a wild-type C. difficile TcdA amino acidsequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,preferably about 98%, more preferably about 99% or most preferably about100% identity to SEQ ID NO: 1 (full length) when optimally aligned, suchas by the programs GAP or BESTFIT using default gap weights.

In a preferred embodiment, the wild-type C. difficile TcdA includes anamino acid sequence set forth in SEQ ID NO: 1, which describes thewild-type amino acid sequence for TcdA from C. difficile strain 630(also disclosed in GenBank accession number YP_(—)001087137.1 and/orCAJ67494.1). C. difficile strain 630 is known in the art as being aPCR-ribotype 012 strain. SEQ ID NO: 9 describes the wild-type gene forTcdA from C. difficile strain 630, which is also disclosed in GenBankaccession number NC_(—)009089.1.

Another example of a wild-type C. difficile TcdA includes an amino acidsequence set forth in SEQ ID NO: 15, which describes the wild-type aminoacid sequence for TcdA from C. difficile strain R20291 (also disclosedin GenBank accession number YP_(—)003217088.1). C. difficile strainR20291 is known in the art as being a hypervirulent strain and aPCR-ribotype 027 strain. The amino acid sequence for TcdA from C.difficile strain R20291 has about 98% identity to SEQ ID NO:1. SEQ IDNO: 16 describes the wild-type gene for TcdA from C. difficile strainR20291, which is also disclosed in GenBank accession number NC 013316.1.

An additional example of a wild-type C. difficile TcdA includes an aminoacid sequence set forth in SEQ ID NO: 17, which describes the wild-typeamino acid sequence for TcdA from C. difficile strain CD196 (alsodisclosed in GenBank accession number CBA61156.1). CD196 is a strainfrom a recent Canadian outbreak, and it is known in the art as aPCR-ribotype 027 strain. The amino acid sequence for TcdA from C.difficile strain CD196 has about 98% identity to SEQ ID NO: 1, and hasabout 100% identity to TcdA from C. difficile strain R20291. SEQ ID NO:18 describes the wild-type gene for TcdA from C. difficile strain CD196,which is also disclosed in GenBank accession number FN538970.1.

Further examples of an amino acid sequence for a wild-type C. difficileTcdA include SEQ ID NO: 19, which describes the wild-type amino acidsequence for TcdA from C. difficile strain VPI10463 (also disclosed inGenBank accession number CAA63564.1). The amino acid sequence for TcdAfrom C. difficile strain VPI10463 has about 100% (99.8%) identity to SEQID NO: 1. SEQ ID NO: 20 describes the wild-type gene for TcdA from C.difficile strain VPI10463, which is also disclosed in GenBank accessionnumber X92982.1.

Additional examples of a wild-type C. difficile TcdA include TcdA fromwild-type C. difficile strains obtainable from the Centers for DiseaseControl and Prevention (CDC, Atlanta, Ga.). The inventors discoveredthat the amino acid sequence of TcdA from wild-type C. difficile strainsobtainable from the CDC include at least about 99.3% to 100% identity,when optimally aligned, to amino acid residues 1 to 821 of SEQ ID NO: 1(TcdA from C. difficile 630). See Table 1.

The inventors also discovered that the amino acid sequence of TcdA fromwild-type C. difficile strains may include at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, to about 100% identity, whenoptimally aligned (e.g., when full, length sequences are optimallyaligned) to SEQ ID NO: 1.

Table 1: wild-type C. difficile strains obtained from CDC and thepercent identity of amino acid residues 1-821 of TcdA from therespective wild-type C. difficile strain to amino acid residues 1-821 ofSEQ ID NO: 1, when optimally aligned.

TABLE 1 Wild-type C. difficile Strains from CDC C. difficile Approximate% Amino Acid Identity Strain ID to Residues 1-821 of SEQ ID NO: 12004111 100 2004118 99.6 2004205 100 2004206 100 2005325 99.3 200535999.6 2006017 100 2007070 100 2007302 100 2007816 99.3 2007838 99.62007886 99.6 2008222 100 2009078 100 2009087 100 2009141 100 200929299.6

Accordingly, in one embodiment, the wild-type C. difficile TcdA aminoacid sequence includes a sequence of at least about 500, 600, 700, or800 contiguous residues, which has at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, ormost preferably about 100% identity to a sequence of equal lengthbetween residues 1 to 900 of SEQ ID NO: 1 when optimally aligned, suchas by the programs GAP or BESTFIT using default gap weights. Examplesinclude strains described above (e.g., R20291, CD196, etc) and thoselisted in Table 1.

In another embodiment, the wild-type C. difficile TcdA amino acidsequence includes a sequence having at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, preferably about 97%, preferably about 98%, morepreferably about 99% or most preferably about 100% identity to anysequence selected from SEQ ID NOs: 87-109 when optimally aligned. SeeTable 1-a.

TABLE 1-a Wild-type C. difficile Strains C. difficile Strain ID Toxin A,SEQ ID NO: 2004013 SEQ ID NO: 87 2004111 SEQ ID NO: 88 2004118 SEQ IDNO: 89 2004205 SEQ ID NO: 90 2004206 SEQ ID NO: 91 2005022 SEQ ID NO: 922005088 SEQ ID NO: 93 2005283 SEQ ID NO: 94 2005325 SEQ ID NO: 952005359 SEQ ID NO: 96 2006017 SEQ ID NO: 97 2006376 N/A 2007070 SEQ IDNO: 98 2007217 SEQ ID NO: 99 2007302 SEQ ID NO: 100 2007816 SEQ ID NO:101 2007838 SEQ ID NO: 102 2007858 SEQ ID NO: 103 2007886 SEQ ID NO: 1042008222 SEQ ID NO: 105 2009078 SEQ ID NO: 106 2009087 SEQ ID NO: 1072009141 SEQ ID NO: 108 2009292 SEQ ID NO: 109 001 SEQ ID NO: 148 002 SEQID NO: 149 003 SEQ ID NO: 150 012 (004) SEQ ID NO: 151 014 SEQ ID NO:134 015 SEQ ID NO: 135 017 020 SEQ ID NO: 136 023 SEQ ID NO: 137 027 SEQID NO: 138 029 SEQ ID NO: 139 046 SEQ ID NO: 140 053 SEQ ID NO: 168 059SEQ ID NO: 178 070 SEQ ID NO: 152 075 SEQ ID NO: 153 077 SEQ ID NO: 154078 SEQ ID NO: 169 081 SEQ ID NO: 155 087 SEQ ID NO: 170 095 SEQ ID NO:171 106 SEQ ID NO: 180 117 SEQ ID NO: 156 126 SEQ ID NO: 172 131 SEQ IDNO: 157 SE844 SEQ ID NO: 196 12087 SEQ ID NO: 197 K14 SEQ ID NO: 198 BI6SEQ ID NO: 199 BI17 SEQ ID NO: 200 CH6230 SEQ ID NO: 201 SE881 SEQ IDNO: 202

The wild-type gene for TcdB has about 7098 nucleotides that encode aprotein with a deduced molecular weight of about 270 kDa, having about2366 amino acids. As used herein, a wild-type C. difficile TcdB includesa C. difficile TcdB from any wild-type C. difficile strain. A wild-typeC. difficile TcdB may include a wild-type amino acid sequence having atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about98%, more preferably about 99% or most preferably about 100% identity toSEQ ID NO: 2 when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights. In a preferred embodiment, thewild-type C. difficile TcdB includes an amino acid sequence set forth inSEQ ID NO: 2, which describes the wild-type amino acid sequence for TcdBfrom C. difficile strain 630 (also disclosed in GenBank accession numberYP_(—)001087135.1 and/or CAJ67492). SEQ ID NO: 10 describes thewild-type gene for TcdB from C. difficile strain 630, which is alsodisclosed in GenBank accession number NC_(—)009089.1.

Another example of a wild-type C. difficile TcdB includes an amino acidsequence set forth in SEQ ID NO: 21, which describes the wild-type aminoacid sequence for TcdB from C. difficile strain R20291 (also disclosedin GenBank accession number YP_(—)003217086.1 and/or CBE02479.1). Theamino acid sequence for TcdB from C. difficile strain R20291 has about92% identity to SEQ ID NO: 2. SEQ ID NO: 22 describes the wild-type genefor TcdB from C. difficile strain R20291, which is also disclosed inGenBank accession number NC 013316.1.

An additional example of a wild-type C. difficile TcdB includes an aminoacid sequence set forth in SEQ ID NO: 23, which describes the wild-typeamino acid sequence for TcdB from C. difficile strain CD196 (alsodisclosed in GenBank accession number YP_(—)003213639.1 and/orCBA61153.1). SEQ ID NO: 24 describes the wild-type gene for TcdB from C.difficile strain CD196, which is also disclosed in GenBank accessionnumber NC_(—)013315.1. The amino acid sequence for TcdB from C.difficile strain CD196 has about 92% identity to SEQ ID NO: 2.

Further examples of an amino acid sequence for a wild-type C. difficileTcdB include SEQ ID NO: 25, which describes the wild-type amino acidsequence for TcdB from C. difficile strain VPI10463 (also disclosed inGenBank accession number P18177 and/or CAA37298). The amino acidsequence for TcdB from C. difficile strain VPI10463 has 100% identity toSEQ ID NO: 2. SEQ ID NO: 26 describes the wild-type gene for TcdB fromC. difficile strain VPI10463, which is also disclosed in GenBankaccession number X53138.1.

Additional examples of a wild-type C. difficile TcdB include TcdB fromwild-type C. difficile strains obtainable from the Centers for DiseaseControl and Prevention (CDC, Atlanta, Ga.). The inventors discoveredthat the amino acid sequence of TcdB from wild-type C. difficile strainsobtainable from the CDC include at least about 96% to 100% identity,when optimally aligned, to amino acid residue 1 to 821 of SEQ ID NO: 2(TcdB from C. difficile 630). See Table 2.

Table 2: wild-type C. difficile strains obtained from CDC and the %identity of amino acid residues 1-821 of TcdB from the respectivewild-type C. difficile strain to amino acid residues 1-821 of SEQ ID NO:2, when optimally aligned.

TABLE 2 Wild-type C. difficile Strains from CDC C. difficile Approximate% Amino Acid Identity Strain ID to Residues 1-821 of SEQ ID NO: 22004013 96.0 2004111 100 2004118 96.0 2004206 100 2005022 100 200532596.7 2007302 100 2007816 96.7 2008222 100 2009078 100 2009087 1002009141 100

Accordingly, in one embodiment, a wild-type C. difficile TcdB amino acidsequence includes a sequence of at least about 500, 600, 700, or 800contiguous residues, which has at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, preferably about 97%, preferably about 98%, more preferablyabout 99% or most preferably about 100% identity to a sequence of equallength between residues 1 to 900 of SEQ ID NO: 2 when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights.Examples include strains described above (e.g., R20291, CD196, etc) andthose listed in Table 2.

In another embodiment, the wild-type C. difficile TcdB amino acidsequence includes a sequence having at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, preferably about 97%, preferably about 98%, morepreferably about 99% or most preferably about 100% identity to anysequence selected from SEQ ID NOs: 110-133 when optimally aligned. SeeTable 2-a.

TABLE 2-a Wild-type C. difficile Strains C. difficile Strain ID Toxin B,SEQ ID NO: 2004013 SEQ ID NO: 110 2004111 SEQ ID NO: 111 2004118 SEQ IDNO: 112 2004205 SEQ ID NO: 113 2004206 SEQ ID NO: 114 2005022 SEQ ID NO:115 2005088 SEQ ID NO: 116 2005283 SEQ ID NO: 117 2005325 SEQ ID NO: 1182005359 SEQ ID NO: 119 2006017 SEQ ID NO: 120 2006376 SEQ ID NO: 1212007070 SEQ ID NO: 122 2007217 SEQ ID NO: 123 2007302 SEQ ID NO: 1242007816 SEQ ID NO: 125 2007838 SEQ ID NO: 126 2007858 SEQ ID NO: 1272007886 SEQ ID NO: 128 2008222 SEQ ID NO: 129 2009078 SEQ ID NO: 1302009087 SEQ ID NO: 131 2009141 SEQ ID NO: 132 2009292 SEQ ID NO: 133 001SEQ ID NO: 158 002 SEQ ID NO: 159 003 SEQ ID NO: 160 012 (004) SEQ IDNO: 161 014 SEQ ID NO: 141 015 SEQ ID NO: 142 017 SEQ ID NO: 182 020 SEQID NO: 143 023 SEQ ID NO: 144 027 SEQ ID NO: 145 029 SEQ ID NO: 146 046SEQ ID NO: 147 053 SEQ ID NO: 173 059 SEQ ID NO: 179 070 SEQ ID NO: 162075 SEQ ID NO: 163 077 SEQ ID NO: 164 078 SEQ ID NO: 174 081 SEQ ID NO:165 087 SEQ ID NO: 175 095 SEQ ID NO: 176 106 SEQ ID NO: 181 117 SEQ IDNO: 166 126 SEQ ID NO: 177 131 SEQ ID NO: 167

The genes for toxins A and B (tcdA and tcdB) are part of a 19.6-kbgenetic locus (the pathogenicity locus, PaLoc) that includes 3additional small open-reading frames (ORFS), tcdD, tcdE, and tcdC, andmay be considered useful for virulence. The PaLoc is known to be stableand conserved in toxigenic strains. It is present at the samechromosomal integration site in all toxigenic strains that have beenanalyzed to date. In nontoxigenic strains, the pathogenicity locus(PaLoc) is not present. Accordingly, a characteristic of the wild-typeC. difficile strains described herein is the presence of a pathogenicitylocus. Another preferred characteristic of the wild-type C. difficilestrains described herein is the production of both TcdA and TcdB.

In one embodiment, the wild-type C. difficile strain is a strain havinga pathogenicity locus that is at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, preferably about 98%, more preferably about 99% or mostpreferably about 100% identical to that of C. difficile 630 or VPI10463.The total pathogenicity locus sequence of C. difficile VPI10463, isregistered at the EMBL database with the sequence accession numberX92982, also shown in SEQ ID NO: 26. Strains in which the PaLoc isidentical to that of the reference strain VPI10463 are referred to astoxinotype 0. Strains of toxinotypes I-VII, IX, XII-XV, and XVIII-XXIVproduce both TcdA and TcdB despite variations in their toxin genes.

At the N-terminus of the toxins, the glucosyltransferase domain islocated. The glucosyltransferase activity of the toxins is associatedwith the cytotoxic function of the toxins. Without being bound bymechanism or theory, the glucosyltransferase activity in both toxins isbelieved to catalyze the monoglucosylation of small GTP-binding proteinsin the Rho/Rac/Ras superfamily. After glucosylation of these GTP bindingproteins, cellular physiology is modified dramatically, resulting in aloss of structural integrity and disruption of essential signalingpathways of the host cells infected by the toxins. The Asp-Xaa-Asp (DXD)motif, which is involved with manganese, uridine diphosphate (UDP), andglucose binding, is a typical characteristic for the glucosyltransferasedomain. Without being bound by mechanism or theory, it is believed thatresidues critical for catalytic activity, such as the DXD motif, do notvary between a TcdB from a known “historical” strain, such as 630, and aTcdB from a hypervirulent strain, such as R20291. The DXD motif islocated at residues 285 to 287 of a wild-type C. difficile TcdA,according to the numbering of SEQ ID NO: 1, and at residues 286 to 288of a wild-type C. difficile TcdB, according to the numbering of SEQ IDNO: 2.

Global alignment algorithms (e.g., sequence analysis programs) are knownin the art and may be used to optimally align two or more amino acidtoxin sequences to determine if the toxin includes a particularsignature motif (e.g., DXD in the glucosyltransferase domain, DHC in thecysteine protease domain described below, etc.). The optimally alignedsequence(s) are compared to a respective reference sequence (e.g., SEQID NO:1 for TcdA or SEQ ID NO: 2 for TcdB) to determine the existence ofthe signature motif. “Optimal alignment” refers to an alignment givingthe highest percent identity score. Such alignment can be performedusing known sequence analysis programs. In one embodiment, a CLUSTALalignment (such as CLUSTALW) under default parameters is used toidentify suitable wild-type toxins by comparing the query sequenceagainst the reference sequence. The relative numbering of the conservedamino acid residues is based on the residue numbering of the referenceamino acid sequence to account for small insertions or deletions (forexample, five amino acids of less) within the aligned sequence.

As used herein, the term “according to the numbering of” refers to thenumbering of the residues of a reference sequence when the given aminoacid or polynucleotide sequence is compared to the reference sequence.In other words, the number or residue position of a given polymer isdesignated with respect to the reference sequence rather than by theactual numerical position of the residue within the given amino acid orpolynucleotide sequence.

For example, a given amino acid sequence, such as that of ahypervirulent wild-type C. difficile strain, can be aligned to areference sequence (e.g., such as that of a historical wild-type C.difficile strain, e.g., 630) by introducing gaps, if necessary, tooptimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned. As used herein, a“reference sequence” refers to a defined sequence used as a basis for asequence comparison.

Unless stated otherwise, all references herein to amino acid positionsof a TcdA refer to the numbering of SEQ ID NO: 1. Unless statedotherwise, all references herein to amino acid positions of a TcdB referto the numbering of SEQ ID NO: 2.

The glucosyltransferase domain of TcdA, as used herein, may begin atexemplary residue 1, 101, or 102, and may end at exemplary residue 542,516, or 293 of a wild-type C. difficile TcdA, e.g., SEQ ID NO: 1. Anyminimum residue position may be combined with a maximum residue positionbetween residues 1 and 542 of TcdA to define a sequence for theglucosyltransferase domain as long as the DXD motif region is included.For example, in one embodiment, the glucosyltransferase domain of TcdAincludes SEQ ID NO: 27, which is identical to residues 101-293 of SEQ IDNO: 1, and it includes the DXD motif region. In another embodiment, theglucosyltransferase domain of TcdA includes SEQ ID NO: 28, which isidentical to residues 1-542 of SEQ ID NO: 1.

The glucosyltransferase domain of TcdB, as used herein, may begin atexemplary residue 1, 101, or 102, and may end at exemplary residue 543,516, or 293 of a wild-type C. difficile TcdB, e.g., SEQ ID NO: 2. Anyminimum residue position may be combined with a maximum residue positionbetween residues 1 and 543 of TcdB to define a sequence for theglucosyltransferase domain as long as the DXD motif region is included.For example, in one embodiment, the glucosyltransferase domain of TcdBincludes SEQ ID NO: 29, which is identical to residues 101-293 of SEQ IDNO: 2, and it includes the DXD motif region. In another embodiment, theglucosyltransferase domain of TcdB includes SEQ ID NO: 30, which isidentical to residues 1-543 of SEQ ID NO: 2.

Without being bound to theory or mechanism, it is believed that theN-terminus of TcdA and/or TcdB is cleaved by an autoproteolytic processfor the glucosyltransferase domain to be translocated and released intothe host cell cytosol, where it can interact with Rac/Ras/Rho GTPases.Wild-type C. difficile TcdA has been shown to be cleaved between L542and S543. Wild-type C. difficile TcdB has been shown to be cleavedbetween L543 and G544.

The cysteine protease domain is associated with the autocatalyticproteolytic activity of the toxin. The cysteine protease domain islocated downstream of the glucosyltransferase domain and may becharacterized by the catalytic triad aspartate, histidine, and cysteine(DHC), e.g., D589, H655, and C700 of a wild-type TcdA, and D587, H653,and C698 of a wild-type TcdB. Without being bound by mechanism ortheory, it is believed that the catalytic triad is conserved between atoxin from a “historical” strain, such as 630, and a TcdB from ahypervirulent strain, such as R20291.

The cysteine protease domain of TcdA, as used herein, may begin atexemplary residue 543, and may end at exemplary residue 809 769, 768, or767 of a wild-type TcdA, e.g., SEQ ID NO: 1. Any minimum residueposition may be combined with a maximum residue position between 543 and809 of a wild-type TcdA to define a sequence for the cysteine proteasedomain as long as the catalytic triad DHC motif region is included. Forexample, in one embodiment, the cysteine protease domain of TcdAincludes SEQ ID NO: 32, which has the DHC motif region located atresidues 47, 113, and 158 of SEQ ID NO: 32, which respectivelycorrespond to D589, H655, and C700 of a wild-type TcdA according to thenumbering of SEQ ID NO: 1. SEQ ID NO: 32 is identical to residues 543 to809 of SEQ ID NO: 1, TcdA.

The cysteine protease domain of TcdB, as used herein, may begin atexemplary residue 544, and may end at exemplary residue 801, 767, 755,or 700 of a wild-type TcdB, e.g., SEQ ID NO: 2. Any minimum residueposition may be combined with a maximum residue position between 544 and801 of a wild-type TcdB to define a sequence for the cysteine proteasedomain as long as the catalytic triad DHC motif region is included. Forexample, in one embodiment, the cysteine protease domain of TcdBincludes SEQ ID NO: 33, which includes the DHC motif region located atresidues 44, 110, and 115 of SEQ ID NO: 33, which respectivelycorrespond to D587, H653, and C698 of a wild-type TcdB according to thenumbering of SEQ ID NO: 2. SEQ ID NO: 33 is identical to residues 544 to767 of SEQ ID NO: 2, TcdB. In another embodiment, the cysteine proteasedomain of TcdB includes residues 544-801 of SEQ ID NO: 2, TcdB.

Mutant Toxin

In the present invention, the immunogenic composition includes a mutantC. difficile toxin. The term “mutant,” as used herein, refers to amolecule that exhibits a structure or sequence that differs from thecorresponding wild-type structure or sequence, e.g., by havingcrosslinks as compared to the corresponding wild-type structure and/orby having at least one mutation, as compared to the correspondingwild-type sequence when optimally aligned, such as by the programs GAPor BESTFIT using default gap weights. The term “mutant” as used hereinfurther includes a molecule that exhibits a functional property (e.g.,abrogated glucosyltransferase and/or abrogated cysteine proteaseactivity) that differs from the corresponding wild-type molecule.

A C. difficile toxin from any of the wild-type strains described abovemay be used as a source from which a mutant C. difficile toxin isproduced. Preferably, C. difficile 630 is the source from which a mutantC. difficile toxin is produced.

The mutation may involve a substitution, deletion, truncation ormodification of the wild type amino acid residue normally located atthat position. Preferably, the mutation is a non-conservative amino acidsubstitution. The present invention also contemplates isolatedpolynucleotides that include nucleic acid sequences encoding any of themutant toxins described herein.

A “non-conservative” amino acid substitution, as used herein, refers toan exchange of an amino acid from one class for an amino acid fromanother class, according to the following Table 3:

TABLE 3 Amino Acid Classes Class Amino acid Nonpolar: Ala (A), Val (V),Leu (L), Ile (I), Pro (P), Met (M), Phe (F), Trp (W) Uncharged polar:Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) Acidic:Asp (D), Glu (E) Basic: Lys (K), Arg (R), His (H)

Examples of a non-conservative amino acid substitution include asubstitution wherein an aspartic acid residue (Asp, D) is replaced by analanine residue (Ala, A). Other examples include replacing an asparticacid residue (Asp, D) with an asparagine residue (Asn, N); replacing anarginine (Arg, R), glutamic acid (Glu, E), lysine (Lys, K), and/orhistidine (His, H) residue with an alanine residue (Ala, A).

A conservative substitution refers to an exchange between amino acidsfrom the same class, for example, according to Table 3.

The mutant toxins of the invention may be prepared by techniques knownin the art for preparing mutations, such as, for example, site-directedmutagenesis, mutagenesis using a mutagen (e.g., UV light), etc.Preferably, site-directed mutagenesis is used. Alternatively, a nucleicacid molecule having an objective sequence may be directly synthesized.Such chemical synthesis methods are known in the art.

In the present invention, the mutant C. difficile toxin includes atleast one mutation in a glucosyltransferase domain, relative to thecorresponding wild-type C. difficile toxin. In one embodiment, theglucosyltransferase domain includes at least two mutations. Preferably,the mutation decreases or abrogates glucosyltransferase enzyme activityof the toxin, as compared to the glucosyltransferase enzyme activity ofthe corresponding wild-type C. difficile toxin.

Exemplary amino acid residues in a glucosyltransferase domain of TcdAthat may undergo a mutation include at least one of the following, orany combination thereof: W101, D269, R272, D285, D287, E460, R462, S541,and L542, as compared to a wild-type C. difficile TcdA, according to thenumbering of SEQ ID NO: 1. Further exemplary amino acid residues thatmay undergo a mutation include E514, S517, and W519, as compared to awild-type C. difficile TcdA, according to the numbering of SEQ ID NO: 1.

Exemplary mutations in a glucosyltransferase domain of TcdA include atleast one of the following, or any combination thereof: W101A, D269A,R272A, D285A, D287A, E460A, R462A, S541A, and L542G, as compared to awild-type C. difficile TcdA. In a preferred embodiment, theglucosyltransferase domain of TcdA includes a L542G mutation, ascompared to a wild-type C. difficile TcdA. In another preferredembodiment, the glucosyltransferase domain of TcdA includes a D285A anda D287A mutation, as compared to a wild-type C. difficile TcdA.

Exemplary amino acid residues in a glucosyltransferase domain of TcdBthat may undergo a mutation include at least one of the following, orany combination thereof: W102, D270, R273, D286, D288, N384, D461, K463,W520, and L543, as compared to a wild-type C. difficile toxin B,according to the numbering of SEQ ID NO: 2. Further exemplary amino acidresidues that may undergo a mutation include E515, S518, and W520, ascompared to a wild-type C. difficile toxin B, according to the numberingof SEQ ID NO: 2.

Exemplary mutations in a glucosyltransferase domain of TcdB include atleast one of the following, or any combination thereof: W102A, D270A,D270N, R273A, D286A, D288A, N384A, D461A, D461R, K463A, K463E, W520A,and L543A, as compared to a wild-type C. difficile TcdB. In a preferredembodiment, the glucosyltransferase domain of TcdB includes a L543A, ascompared to a wild-type C. difficile TcdB. In another preferredembodiment, the glucosyltransferase domain of TcdB includes a D286A anda D288A mutation, as compared to a wild-type C. difficile TcdB.

Any of the mutations described herein above may be combined with amutation in a cysteine protease domain. In the present invention, themutant C. difficile toxin includes at least one mutation in a cysteineprotease domain, relative to the corresponding wild-type C. difficiletoxin. Preferably, the mutation decreases or abrogates cysteine proteaseactivity of the toxin, as compared to the cysteine protease activity ofthe corresponding wild-type C. difficile toxin.

Exemplary amino acid residues in a cysteine protease domain of TcdA thatmay undergo a mutation include at least one of the following, or anycombination thereof: S543, D589, H655, and C700, as compared to awild-type C. difficile TcdA, according to the numbering of SEQ ID NO: 1.Exemplary mutations in a glucosyltransferase domain of TcdA include atleast one of the following, or any combination thereof: S543A, D589A,D589N, H655A, C700A, as compared to a wild-type C. difficile TcdA. In apreferred embodiment, the cysteine protease domain of TcdA includes aC700A mutation, as compared to a wild-type C. difficile TcdA.

Exemplary amino acid residues in a cysteine protease domain of TcdB thatmay undergo a mutation include at least one of the following, or anycombination thereof: G544, D587, H653, and C698, as compared to awild-type C. difficile TcdB, according to the numbering of SEQ ID NO: 2.Exemplary mutations in a glucosyltransferase domain of TcdB include atleast one of the following, or any combination thereof: G544A, D587A,D587N, H653A, C698A, as compared to a wild-type C. difficile TcdB. In apreferred embodiment, the cysteine protease domain of TcdB includes aC698A mutation, as compared to a wild-type C. difficile TcdB. Additionalamino acid residues in a cysteine protease domain of TcdB that mayundergo a mutation include: K600 and/or R751, as compared to a wild-typeTcdB. Exemplary mutations include K600E and/or R751E.

Accordingly, the inventive mutant C. difficile toxin includes aglucosyltransferase domain having a mutation and a cysteine proteasedomain having a mutation, relative to the corresponding wild-type C.difficile toxin. In one embodiment, the mutant toxin includes at leastone mutation in the glucosyltransferase domain and at least one mutationin the cysteine protease domain. In a preferred embodiment, a mutanttoxin A includes at least a D285, D287, and C700 mutation. In apreferred embodiment, a mutant toxin B includes at least a D286, D288,and C698 mutation. The mutant toxins may include any further mutation,individually or in combination, described herein.

An exemplary mutant C. difficile TcdA includes a glucosyltransferasedomain including SEQ ID NO: 29 having an amino acid substitution atpositions 285 and 287, and a cysteine protease domain comprising SEQ IDNO: 32 having an amino acid substitution at position 158, relative tothe corresponding wild-type C. difficile toxin A. For example, such amutant C. difficile TcdA includes the amino acid sequence set forth inSEQ ID NO: 4, wherein the initial methionine is optionally not present.In another embodiment, the mutant mutant C. difficile toxin A includesthe amino acid sequence set forth in SEQ ID NO: 84.

Further examples of a mutant C. difficile toxin A include the amino acidsequence set forth in SEQ ID NO: 7, which has a D269A, R272A, D285A,D287A, E460A, R462A, and C700A mutation, as compared to SEQ ID NO: 1,wherein the initial methionine is optionally not present. In anotherembodiment, the mutant mutant C. difficile toxin A includes the aminoacid sequence set forth in SEQ ID NO: 83.

Another exemplary mutant TcdA includes SEQ ID NO: 34, wherein theresidue at positions 101, 269, 272, 285, 287, 460, 462, 541, 542, 543,589, 655, and 700 may be any amino acid.

In some embodiments, the mutant C. difficile toxin exhibits decreased orabrogated autoproteolytic processing as compared to the correspondingwild-type C. difficile toxin. For example, a mutant C. difficile TcdAmay include a mutation at one of the following residues, or anycombination thereof: S541, L542 and/or S543, as compared to thecorresponding wild-type C. difficile TcdA. Preferably, the mutant C.difficile TcdA includes at least one of the following mutations, or anycombination thereof: S541A, L542G, and S543A, as compared to thecorresponding wild-type C. difficile TcdA.

Another exemplary mutant C. difficile TcdA includes a S541A, L542, S543and C700 mutation, as compared to the corresponding wild-type C.difficile TcdA.

An exemplary mutant C. difficile toxin B includes a glucosyltransferasedomain comprising SEQ ID NO: 31 having an amino acid substitution atpositions 286 and 288, and a cysteine protease domain comprising SEQ IDNO: 33 having an amino acid substitution at position 155, relative tothe corresponding wild-type C. difficile toxin B. For example, such amutant C. difficile TcdB includes the amino acid sequence set forth inSEQ ID NO: 6, wherein the initial methionine is optionally not present.In another embodiment, the mutant mutant C. difficile toxin A includesthe amino acid sequence set forth in SEQ ID NO: 86.

Further examples of a mutant C. difficile TcdB include the amino acidsequence set forth in SEQ ID NO: 8, which has a D270A, R273A, D286A,D288A, D461A, K463A, and C698A mutation, as compared to SEQ ID NO: 2.SEQ ID NO: 8 wherein the initial methionine is optionally not present.In another embodiment, the mutant mutant C. difficile toxin A includesthe amino acid sequence set forth in SEQ ID NO: 85.

Another exemplary mutant TcdB includes SEQ ID NO: 35, wherein theresidue at positions 101, 269, 272, 285, 287, 460, 462, 541, 542, 543,589, 655, and 700 may be any amino acid.

As another example, a mutant C. difficile TcdB may include a mutation atpositions 543 and/or 544, as compared to the corresponding wild-type C.difficile TcdB. Preferably, the mutant C. difficile TcdB includes a L543and/or G544 mutation, as compared to the corresponding wild-type C.difficile TcdB. More preferably, the mutant C. difficile TcdB includes aL543G and/or G544A mutation, as compared to the corresponding wild-typeC. difficile TcdB.

Another exemplary mutant C. difficile TcdB includes a L543G, G544A andC698 mutation, as compared to the corresponding wild-type C. difficileTcdB.

In one aspect, the invention relates to an isolated polypeptide having amutation at any position from amino acid residue 1 to 1500 according tothe numbering of SEQ ID NO: 2, to define an exemplary mutant C.difficile toxin B. For example, in one embodiment, the isolatedpolypeptide includes a mutation between amino acids residues 830 and 990of SEQ ID NO: 2. Exemplary positions for mutations include positions 970and 976 according to the numbering of SEQ ID NO: 2. Preferably, themutation between residues 830 and 990 is a substitution. In oneembodiment, the mutation is a non-conservative substitution wherein anAsp (D) and/or a Glu (E) amino acid residue is replaced by an amino acidresidue that is not neutralized upon acidification, such as, forexample, lysine (K), arginine (R), and histidine (H). Exemplarymutations include: E970K, E970R, E970H, E976K, E976R, E976H of SEQ IDNO: 2, to define a mutant C. difficile toxin B.

In one embodiment, the isolated polypeptide includes the followingsubstitutions D286A/D288A/C698A/E970K/E976K (SEQ ID NO: 184). E970 andE976 are conserved in toxin B from all C. difficile strains observed(see Table 2-a, SEQ ID NOs: 110-133), except in ribotype 078 andribotype 126 strains (see Table 35 and Table 37). In toxin B fromribotype 078 and ribotype 126 strains, there is a glycine-970 (G970)instead of glutamate-970. Accordingly, in one embodiment, the isolatedpolypeptide includes a mutation at G970 and E976, such as, for example,G970K and E976K. The mutant toxins described above and herein mayexhibit reduced cytotoxicity as compared to the corresponding wild-typetoxin. See Examples 8 and 15).

In another aspect, the invention relates to an isolated polypeptidehaving a mutation at any position from amino acid residue 1 to 1500according to the numbering of SEQ ID NO: 1, to define an exemplarymutant C. difficile toxin A. For example, in one embodiment, theisolated polypeptide includes a mutation between amino acids residues832 and 992 of SEQ ID NO: 1. Exemplary positions for mutations includepositions 972 and 978 according to the numbering of SEQ ID NO: 1.Preferably, the mutation between residues 832 and 992 is a substitution.In one embodiment, the mutation is a non-conservative substitutionwherein an Asp (D) and/or a Glu (E) amino acid residue is replaced by anamino acid residue that is not neutralized upon acidification, such as,for example, lysine (K), arginine (R), and histidine (H). Exemplarymutations include: D972K, D972R, D972H, D978K, D978R, D978H of SEQ IDNO: 1, to define a mutant C. difficile toxin A.

In one embodiment, the isolated polypeptide includes the followingsubstitutions D285A/D287A/C700A/D972K/D978K (SEQ ID NO: 183). D972 andD978 residues are conserved in toxin A from all C. difficile strainsassessed (see Table 1-a, SEQ ID NOs: 87-109). The mutant toxinsdescribed above and herein may exhibit reduced cytotoxicity as comparedto the corresponding wild-type toxin.

The following describes additional exemplary mutant toxins. In oneembodiment, the mutant toxin TcdA includes (i) SEQ ID NO: 185; (ii) apolypeptide of SEQ ID NO:185 having at least 90%, 92%, 93%, 95%, 98%,99% or 100% identity to SEQ ID NO:185; or (iii) a fragment of at least250, 280 or 300 amino acids of SEQ ID NO:185. In another embodiment, themutant toxin TcdB includes (iv) SEQ ID NO:186; (v) a polypeptide of SEQID NO:186 having at least 80%, 85%, 90%, 92%, 93%, 95%, 98%, 99% or 100%identity to SEQ ID NO:186; or (vi) a fragment of at least 250, 280 or300 amino acids of SEQ ID NO:186.

In one embodiment, the mutant toxin TcdA consists of less than 600, 675,650, 625, 600, 575, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325,300, 275, 250, or 225 amino acids. In one embodiment, the mutant toxinconsists of less than 800, 775, 750, 725, 700, 675, 650, 625, 600, 575,550, or 525 amino acids. In one embodiment, the mutant toxin includes atleast 200, 225, 250, 270, 280, 300 or 310 amino acids of SEQ ID NO:185or at least 200, 225, 250, 270, 280, 300 or 310 amino acids of apolypeptide having at least 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100%identity to SEQ ID NO:185. In one embodiment the mutant toxin includesat least 400, 425, 450, 475, 500, 525, 550, 575, 600 or 610 amino acidsof SEQ ID NO: 186 or at least 400, 425, 450, 475, 500, 525, 550, 575,600 or 610 amino acids of a polypeptide having at least 80%, 85%, 90%,92%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:186.

In one embodiment, the mutant toxin includes a fusion protein thatincludes A) (i) SEQ ID NO:185; (ii) a polypeptide of SEQ ID NO:185having at least 90%, 92%, 93%, 95%, 98%, 99% or 100% identity to SEQ IDNO:185; or (iii) a fragment of at least 250, 280 or 300 amino acids ofSEQ ID NO:185 fused to B) (iv) SEQ ID NO:186; (v) a polypeptide of SEQID NO:186 having at least 80%, 85%, 90%, 92%, 93%, 95%, 98%, 99% or 100%identity to SEQ ID NO:186; or (vi) a fragment of at least 250, 280 or300 amino acids of SEQ ID NO:186. In a further embodiment the N-terminusof SEQ ID NO: 185 is adjacent to the C-terminus of SEQ ID NO: 186. In afurther embodiment the N-terminus of SEQ ID NO: 185 is adjacent to theN-terminus of SEQ ID NO: 186. In a further embodiment the C-terminus ofSEQ ID NO:185 is adjacent to the C-terminus of SEQ ID NO:186. Furtherexamples of a mutant toxin include a polypeptide having any one of thefollowing amino acid sequences SEQ ID NO: 187, SEQ ID NO: 188, SEQ IDNO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193,SEQ ID NO: 194, and SEQ ID NO: 195.

In another embodiment, the mutant toxin includes a fusion and/or hybridpolypeptide that includes any combination of amino acid sequencesselected from any of the following: SEQ ID NO: 223, SEQ ID NO: 224, SEQID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO:229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236. SEQ ID NO: 237, SEQ ID NO:238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, andSEQ ID NO: 243. For example, in one embodiment, the mutant toxinincludes an amino acid sequence as set forth in any one of thefollowing: SEQ ID NO: 254, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO:272, SEQ ID NO: 273, SEQ ID NO: 274, or SEQ ID NO: 275.

Another example of a mutant toxin includes a fragment of a wild-typetoxin. A “fragment” mutant toxin TcdA as used herein refers to a peptidesequence that has less consecutive amino acids total than thecorresponding wild-type C. difficile toxin TcdA sequence, e.g., asequence that includes less than 2710 consecutive amino acids total. Thefragment mutant toxin TcdA may further include a mutation of an aminoacid residue as described herein. A “fragment” mutant toxin TcdB as usedherein refers to a peptide sequence that has less consecutive aminoacids total than the corresponding wild-type C. difficile toxin TcdBsequence, e.g., a sequence that includes less than 2366 consecutiveamino acids total. The fragment mutant toxin TcdB may further include amutation of an amino acid residue as described herein. Such exemplarymutant toxin sequences include SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33. In one embodiment, themutant toxin TcdA includes at least 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000consecutive amino acids of a wild-type mutant toxin A, as describedherein, e.g., SEQ ID NO: 1. In another embodiment, a mutant toxinincludes a fragment of a genetically mutated toxin A described herein.In one embodiment, the mutant toxin TcdB includes at least 200, 300,400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, or 2000 consecutive amino acids of a wild-type mutanttoxin B, as described herein, e.g., SEQ ID NO: 2. In another embodiment,a mutant toxin includes a fragment of a genetically mutated toxin Bdescribed herein. In one embodiment, the mutant toxin includes at most3000, 2710, 2500, 2400, 2366, 2000, 1900, 1800, 1700, 1600, 1500, 1400,1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, or 300 consecutiveamino acids. Any minimum value may be combined with any maximum value todefine a suitable range. In another embodiment, the mutant toxin isfused to at least one other peptide or at least one other mutant toxin amanner that results in the production of a hybrid molecule.

Additional exemplary fragment mutant toxin TcdA includes a polypeptidehaving any one of the amino acid sequences of SEQ ID NO: 374 through SEQID NO: 421. Further exemplary fragment mutant toxin TcdA includes apolypeptide having any one of the amino acid sequences of SEQ ID NO: 472through SEQ ID NO: 519. Additional exemplary mutant toxin TcdB includesa polypeptide having any one of the amino acid sequences of SEQ ID NO:422 through SEQ ID NO: 471. Further exemplary fragment mutant toxin TcdBincludes a polypeptide having any one of the amino acid sequences of SEQID NO: 568 through SEQ ID NO: 615.

The following describes further exemplary mutant toxins. In oneembodiment, the mutant toxin includes a TcdA which includes or consistsof at least 3, at least 4, or at least 5 mutations at amino acidresidues selected from the group consisting of: W101, D287, E514, D285,S517, W519, and C700, e.g., according to the numbering of SEQ ID NO: 1.In additional embodiments; the TcdA mutants include or consist of atleast 3, at least 4, or at least 5 mutations selected from the groupconsisting of: W101A, D287A, E514Q, D285A, S517A, W519A, and C700Asubstitutions and a W101 deletion.

Another exemplary mutant toxin TcdA includes the amino acidsubstitutions W101, D287A, and E514Q, e.g., according to the numberingof SEQ ID NO: 1. A further embodiment provides a TcdA protein includingor consisting of the amino acid substitutions W101A, D287A, E514Q, andW519A. Another specific embodiment of the invention is a TcdA proteinincluding or consisting of the amino acid substitutions W101A, D287A,E514Q, W519A, and C700A.

In another embodiment, the mutant toxin TcdA includes the mutationsW101A, D287A, E514Q and D285A, e.g., according to the numbering of SEQID NO: 1. In another embodiment, the mutant toxin TcdA includes themutations W101A, D287A, E514Q and S517A.

In another embodiment, a further mutation may be added to the mutantTcdA, e.g. a C700A mutation. In additional embodiments, up to 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 furthermutations may be added to any of the mutant toxin TcdA embodimentsdescribed herein.

Further examples of a mutant toxin TcdA includes the amino acid sequenceas set forth in SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ IDNO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, or SEQ ID NO:211. In another embodiment, the mutant toxin TcdA includes the aminoacid sequence SEQ ID NO: 210. In yet another embodiment, the mutanttoxin TcdA includes a mutation at positions W101, D287, E514, W519 andC700; wherein W101 is replaced with any amino acid except tryptophan,D287 is replaced with any amino acid but aspartic acid, E514 is replacedwith any amino acid but glutamic acid, W519 is replaced with any aminoacid but tryptophan and C700 is replaced with any amino acid butcysteine, as set forth in SEQ ID NO: 212.

Additional examples of a mutant toxin TcdA include a polypeptide havingan amino acid sequence that is at least 85%, at least 90%, at least 95%,at least 97%, at least 98%, or at (east 99% identical to the originalreference sequence (e.g. SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205,SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ IDNO: 210, SEQ ID NO: 211 or SEQ ID NO: 212 or SEQ ID NO: 82, SEQ ID NO:83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86).

In a further embodiment, mutant toxin TcdA includes mutations at at mostthan 12 amino acid residues, at most than 11 amino acid residues, atmost than 10 amino acid residues, at most than 9 amino acid residues, atmost than 8 amino acid residues, at most than 7 amino acid residues, atmost than 6 amino acid residues, at most than 5 amino acid residues, atmost than 4 amino acid residues, at most than 3 amino acid residues, atmost than 2 amino acid residues, or 1 amino acid residue, for example,relative to SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQID NO: 211 and SEQ ID NO: 212 or SEQ ID NO: 82, SEQ ID NO: 83, SEQ IDNO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.

The following describes further exemplary mutant toxins. In oneembodiment, the mutant toxin is a mutant TcdB that that includes atleast 3, at least 4, or at least 5 mutations at amino acid residuesselected from the group consisting of: W102, D288, E515, D286, S518,W520, and C698, according to the numbering of SEQ ID NO: 2. In anotherembodiment, the mutant toxin TcdB includes at least 3, at least 4, or atleast 5 mutations selected from the group consisting of: W102A, D288A,E515Q, D286A, S518A, W520A, and C698A substitutions and a W102 deletion.In one embodiment, the mutant toxin TcdB includes the amino acidsequence as set forth in SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215,SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ IDNO: 220, or SEQ ID NO: 221. In yet another embodiment, the mutant toxinTcdB includes a mutation at positions W102, D288, E515, W520 and C698;wherein W102 is replaced with any amino acid except tryptophan, D288 isreplaced with any amino acid but aspartic acid, E515 is replaced withany amino acid but glutamic acid, W520 is replaced with any amino acidbut tryptophan and C698 is replaced with any amino acid but cysteine, asset forth in SEQ ID NO: 222.

Additional examples of a mutant toxin TcdB include a polypeptide havingan amino acid sequence that is at least 85%, at least 90%, at least 95%,at least 97%, at least 98%, or at (east 99% identical to the originalreference sequence SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO:220, SEQ ID NO: 221, or SEQ ID NO: 222.

In a further embodiment, mutant toxin TcdB includes mutations at at mostthan 12 amino acid residues, at most than 11 amino acid residues, atmost than 10 amino acid residues, at most than 9 amino acid residues, atmost than 8 amino acid residues, at most than 7 amino acid residues, atmost than 6 amino acid residues, at most than 5 amino acid residues, atmost than 4 amino acid residues, at most than 3 amino acid residues, atmost than 2 amino acid residues, or 1 amino acid residue, for example,relative to SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO:216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221, and/or SEQ ID NO: 222.

Additional exemplary mutant toxin TcdA includes a polypeptide having anyone of the amino acid sequences of SEQ ID NO: 276 through SEQ ID NO:323. Additional exemplary mutant toxin TcdB includes a polypeptidehaving any one of the amino acid sequences of SEQ ID NO: 324 through SEQID NO: 373.

The following describes further exemplary mutant toxins. In oneembodiment, the mutant toxin TcdA includes an amino acid sequence: (a)having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, 99.9%,or 100%) to SEQ ID NO: 224 or SEQ ID NO: 245; and/or b) that is afragment of at least “n” consecutive amino acids of SEQ ID NO: 224 orSEQ ID NO: 245, wherein “n” is 7 or more (e.g. 8, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, 300, 400,500, 540, or more). or of a polypeptide having 80% or more identity toSEQ ID NO: 224 or SEQ ID NO: 245 and that comprises an epitope of SEQ IDNO: 224 and/or SEQ ID NO: 245.

In one embodiment, the mutant toxin TcdA includes the amino acidsequence set forth in SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225,SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ IDNO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234,SEQ ID NO: 235, or SEQ ID NO: 236. Additional exemplary embodiments of amutant toxin TcdA includes the amino acid sequence set forth in any ofthe following: SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 250, SEQ IDNO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255,SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ IDNO: 260, SEQ ID NO: 261, SEQ ID NO: 262, and/or SEQ ID NO: 263.

In one embodiment, the mutant toxin TcdB includes an amino acid sequence(a) having 80% or more identity to SEQ ID NO: 238 or SEQ ID NO: 247;and/or b) that is a fragment of at least 7 consecutive amino acids ofSEQ ID NO: 238 or SEQ ID NO: 247, or of a polypeptide having 80% or moreidentity to SEQ ID NO: 238 or SEQ ID NO: 247 and that comprises anepitope of SEQ ID NO: 238 or SEQ ID NO: 247.

In one embodiment, the mutant toxin TcdB includes an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%,99.8%, 99.9%, or more) to SEQ ID NO: 238 or SEQ ID NO: 247; and/or (b)that is a fragment of at least “n” consecutive amino acids of SEQ ID NO:238 or SEQ ID NO: 247, or of a polypeptide having 50% or more identityto SEQ ID NO: 238 or SEQ ID NO: 247, wherein “n” is 7 or more (e.g. 8,10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,150, 250, 300, 400, 500, 540, or more).

In one embodiment, the mutant toxin TcdB includes the amino acidsequence set forth in SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239,SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 243.Additional exemplary embodiments of a mutant toxin TcdB includes theamino acid sequence set forth in any of the following: SEQ ID NO: 246,SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 264, SEQ IDNO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, and/or SEQ IDNO: 269.

Additional exemplary mutant toxin TcdA includes a polypeptide having anyone of the amino acid sequences of SEQ ID NO: 520 through SEQ ID NO:567. Additional exemplary mutant toxin TcdB includes a polypeptidehaving any one of the amino acid sequences of SEQ ID NO: 616 through SEQID NO: 663.

The following describes yet additional exemplary mutant toxins. In oneembodiment, the mutant toxin TcdA includes a polypeptide having any oneof the amino acid sequences of SEQ ID NO: 664 through SEQ ID NO: 711.Additional exemplary mutant toxin TcdB includes a polypeptide having anyone of the amino acid sequences of SEQ ID NO: 712 through SEQ ID NO:761.

The polypeptides of the invention may include an initial methionineresidue, in some cases as a result of a host cell-mediated process.Depending on, for example, the host cell used in a recombinantproduction procedure and/or the fermentation or growth conditions of thehost cell, it is known in the art that the N-terminal methionine encodedby the translation initiation codon may be removed from a polypeptideafter translation in cells or the N-terminal methionine may remainpresent in the isolated polypeptide.

Accordingly, in one aspect, the invention relates to an isolatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 4,wherein the initial methionine (at position 1) is optionally notpresent. In one embodiment, the initial methionine of SEQ ID NO: 4 isabsent. In one aspect, the invention relates to an isolated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 84, which isidentical to SEQ ID NO: 4, but for an absence of the initial methionine.

In another aspect, the isolated polypeptide includes the amino acidsequence set forth in SEQ ID NO: 6, wherein the initial methionine (atposition 1) is optionally not present. In one embodiment, the initialmethionine of SEQ ID NO: 6 is absent. In one aspect, the inventionrelates to an isolated polypeptide including the amino acid sequence setforth in SEQ ID NO: 86, which is identical to SEQ ID NO: 6, but for anabsence of the initial methionine.

In a further aspect, the isolated polypeptide includes the amino acidsequence set forth in SEQ ID NO: 7, wherein the initial methionine (atposition 1) is optionally not present. In one embodiment, the inventionrelates to an isolated polypeptide including the amino acid sequence setforth in SEQ ID NO: 83, which is identical to SEQ ID NO: 7, but for anabsence of the initial methionine. In yet another aspect, the isolatedpolypeptide includes the amino acid sequence set forth in SEQ ID NO: 8,wherein the initial methionine (at position 1) is optionally notpresent. In one embodiment, the isolated polypeptide includes the aminoacid sequence set forth in SEQ ID NO: 85, which is identical to SEQ IDNO: 8, but for an absence of the initial methionine.

In one aspect, the invention relates to an immunogenic compositionincluding SEQ ID NO: 4, wherein the initial methionine (at position 1)is optionally not present. In another aspect, the invention relates toan immunogenic composition including SEQ ID NO: 6, wherein the initialmethionine (at position 1) is optionally not present. In a furtheraspect, the invention relates to an immunogenic composition includingSEQ ID NO: 7, wherein the initial methionine (at position 1) isoptionally not present. In yet another aspect, the invention relates toan immunogenic composition including SEQ ID NO: 8, wherein the initialmethionine (at position 1) is optionally not present.

In another aspect, the invention relates to an immunogenic compositionincluding SEQ ID NO: 83. In one aspect, the invention relates to animmunogenic composition including SEQ ID NO: 84. In one aspect, theinvention relates to an immunogenic composition including SEQ ID NO: 85.In another aspect, the invention relates to an immunogenic compositionincluding SEQ ID NO: 86.

Cytotoxicity

In addition to generating an immune response in a mammal, theimmunogenic compositions described herein also have reduced cytotoxicitycompared to the corresponding wild-type C. difficile toxin. Preferably,the immunogenic compositions are safe and have minimal (e.g., about a6-8 log₁₀ reduction) to no cytotoxicity, relative to the cytotoxicity ofa respective wild-type toxin, for administration in mammals.

As used herein, the term cytotoxicity is a term understood in the artand refers to apoptotic cell death and/or a state in which one or moreusual biochemical or biological functions of a cell are aberrantlycompromised, as compared to an identical cell under identical conditionsbut in the absence of the cytotoxic agent. Toxicity can be quantitated,for example, in cells or in mammals as the amount of an agent needed toinduce 50% cell death (i.e., EC₅₀ or ED₅₀, respectively) or by othermethods known in the art.

Assays for indicating cytotoxicity are known in the art, such as cellrounding assays (see, for example, Kuehne et al. Nature. 2010 Oct. 7;467(7316):711-3). The action of TcdA and TcdB causes cells to round(e.g., lose morphology) and die, and such a phenomenon is visible bylight microscopy. See, for example, FIG. 9.

Additional exemplary cytotoxicity assays known in the art includeglucosylation assays relating to phosphor imaging of Ras labeled with[¹⁴C]glucose assays (as described in Busch et al., J Biol Chem. 1998Jul. 31; 273(31):19566-72), and preferably the in vitro cytotoxicityassay described in the Examples below wherein EC₅₀ may refer to aconcentration of an immunogenic composition that exhibits at least about50% of cytopathogenic effect (CPE) in a cell, preferably a human diploidfibroblast cell (e.g., IMR90 cell (ATCC CCL-186™), as compared to anidentical cell under identical conditions in the absence of the toxin.The in vitro cytotoxicity assay may also be used to assess theconcentration of a composition that inhibits at least about 50% of awild-type C. difficile toxin-induced cytopathogenic effect (CPE) in acell, preferably a human diploid fibroblast cell (e.g., IMR90 cell (ATCCCCL-186™), as compared to an identical cell under identical conditionsin the absence of the toxin. Additional exemplary cytotoxicity assaysinclude those described in Doern et al., J Clin Microbiol. 1992 August;30(8):2042-6. Cytotoxicity can also be determined by measuring ATPlevels in cells treated with toxin. For example, a luciferase basedsubstrate such as CELLTITERGLO® (Promega) may be used, which emitsluminescence measured as a relative light unit (RLU). In such an assay,cell viability may be directly proportional to the amount of ATP in thecells or the RLU values.

In one embodiment, the cytotoxicity of the immunogenic composition isreduced by at least about 1000, 2000, 3000, 4000, 5000-, 6000-, 7000-,8000-, 9000-, 10000-, 11000-, 12000-, 13000-fold, 14000-fold,15000-fold, or more, as compared to the corresponding wild-type C.difficile toxin. See, for example, Table 20.

In another embodiment, the cytotoxicity of the immunogenic compositionis reduced by at least about 2-log₁₀, more preferably by about 3-log₁₀,and most preferably by about 4-log₁₀ or more, relative to thecorresponding wild-type toxin under identical conditions. For example, amutant C. difficile TcdB may have an EC₅₀ value of about 10⁻⁹ g/ml asmeasured in a standard cytopathic effect assay (CPE), as compared to anexemplary wild-type C. difficile TcdB which may have an EC₅₀ value of atleast about 10⁻¹² g/ml. See, for example, Tables 7A, 7B, 8A and 8B inthe Examples section below.

In yet another embodiment, the cytotoxicity of the mutant C. difficiletoxin has an EC₅₀ of at least about 50 μg/ml, 100 μg/ml, 200 μg/ml, 300μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml,1000 μg/ml or greater, as measured by, for example, an in vitrocytotoxicity assay, such as one described herein. Accordingly, in apreferred embodiment, the immunogenic compositions and mutant toxins arebiologically safe for administration to mammals.

Without being bound by mechanism or theory, a TcdA having a D285 andD287 mutation, as compared to a wild-type TcdA, and a TcdB having a D286and a D288 mutation, as compared to a wild-type TcdB, were expected tobe defective in glycosyltransferase activity and therefore defective ininducing a cytopathic effect. In addition, a toxin having a mutation inthe DHC motif was expected to be defective in autocatalytic processing,and therefore be without any cytotoxic effects.

However, the inventors surprisingly discovered, among other things, thatexemplary mutant TcdA having SEQ ID NO: 4 and exemplary mutant TcdBhaving SEQ ID NO: 6 unexpectedly exhibited cytotoxicity (albeitsignificantly reduced from wild-type C. difficile 630 toxins) despiteexhibiting dysfunctional glucosyltransferase activity and dysfunctionalcysteine protease activity. Without being bound by mechanism or theory,the mutant toxins are believed to effect cytotoxicity through a novelmechanism. Nevertheless, the exemplary mutant TcdA having SEQ ID NO: 4and exemplary mutant TcdB having SEQ ID NO: 6 were surprisinglyimmunogenic. See Examples below.

Crosslinking

Although chemical crosslinking of a wild-type toxin has a potential tofail in inactivating the toxin, the inventors further discovered thatchemically crosslinking at least one amino acid of a mutant toxinfurther reduced cytotoxicity of the mutant toxin, relative to anidentical mutant toxin lacking chemical crosslinks, and relative to thecorresponding wild-type toxin. Preferably, the mutant toxin is purifiedbefore contact with the chemical crosslinking agent.

Moreover, despite a potential of chemical crosslinking agents to alteruseful epitopes, the inventors surprisingly discovered that agenetically modified mutant C. difficile toxin having at least one aminoacid chemically crosslinked resulted in immunogenic compositions thatelicited multiple neutralizing antibodies or binding fragments thereof.Accordingly, epitopes associated with neutralizing antibody moleculeswere unexpectedly retained following chemical crosslinking.

Crosslinking (also referred to as “chemical inactivation” or“inactivation” herein) is a process of chemically joining two or moremolecules by a covalent bond. The terms “crosslinking reagents,”“crosslinking agents,” and “crosslinkers” refer to molecules that arecapable of reacting with and/or chemically attaching to specificfunctional groups (primary amines, sulhydryls, carboxyls, carbonyls,etc) on peptides, polypeptides, and/or proteins. In one embodiment, themolecule may contain two or more reactive ends that are capable ofreacting with and/or chemically attaching to specific functional groups(primary amines, sulhydryls, carboxyls, carbonyls, etc) on peptides,polypeptides, and/or proteins. Preferably, the chemical crosslinkingagent is water-soluble. In another preferred embodiment, the chemicalcrosslinking agent is a heterobifunctional crosslinker. In anotherembodiment, the chemical crosslinking agent is not a bifunctionalcrosslinker. Chemical crosslinking agents are known in the art.

In a preferred embodiment, the crosslinking agent is a zero-lengthcrosslinking agent. A “zero-length” crosslinker refers to a crosslinkingagent that will mediate or produce a direct crosslink between functionalgroups of two molecules. For example, in the crosslinking of twopolypeptides, a zero-length crosslinker will result in the formation ofa bridge, or a crosslink between a carboxyl group from an amino acidside chain of one polypeptide, and an amino group of anotherpolypeptide, without integrating extrinsic matter. Zero-lengthcrosslinking agents can catalyze, for example, the formation of esterlinkages between hydroxyl and carboxyl moieties, and/or the formation ofamide bonds between carboxyl and primary amino moieties.

Exemplary suitable chemical crosslinking agents include formaldehyde;formalin; acetaldehyde; propionaldehyde; water-soluble carbodiimides(RN═C═NR′), which include 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide(EDC), 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide Hydrochloride,1-Cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimidemetho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), andN,N′-diisopropylcarbodiimide (DIC), and derivatives thereof; andN-hydroxysuccinimide (NHS); phenylglyoxal; and/or UDP-dialdehyde.

Preferably, the crosslinking agent is EDC. When a mutant C. difficiletoxin polypeptide is chemically modified by EDC (e.g., by contacting thepolypeptide with EDC), in one embodiment, the polypeptide includes (a)at least one crosslink between a side chain of an aspartic acid residueof the polypeptide and a side chain of a lysine residue of thepolypeptide. In one embodiment, the polypeptide includes (b) at leastone crosslink between a side chain of a glutamic acid residue of thepolypeptide and a side chain of a lysine residue of the polypeptide. Inone embodiment, the polypeptide includes (c) at least one crosslinkbetween the carboxyl group at the C-terminus of the polypeptide and theamino group of the N-terminus of the polypeptide. In one embodiment, thepolypeptide includes (d) at least one crosslink between the carboxylgroup at the C-terminus of the polypeptide and a side chain of a lysineresidue of the polypeptide. In one embodiment, the polypeptide includes(e) at least one crosslink between a side chain of an aspartic acidresidue of the polypeptide and a side chain of a lysine residue of asecond isolated polypeptide. In one embodiment, the polypeptide includes(f) at least one crosslink between a side chain of a glutamic acidresidue of the polypeptide and a side chain of a lysine residue of asecond isolated polypeptide. In one embodiment, the polypeptide includes(g) at least one crosslink between the carboxyl group at the C-terminusof the polypeptide and the amino group of the N-terminus of a secondisolated polypeptide. In one embodiment, the polypeptide includes (h) atleast one crosslink between the carboxyl group at the C-terminus of thepolypeptide and a side chain of a lysine residue of a second isolatedpolypeptide. See, for example, FIG. 24 and FIG. 25.

The “second isolated polypeptide” refers to any isolated polypeptidethat is present during the reaction with EDC. In one embodiment, thesecond isolated polypeptide is a mutant C. difficile toxin polypeptidehaving an identical sequence as the first isolated polypeptide. Inanother embodiment, the second isolated polypeptide is a mutant C.difficile toxin polypeptide having a different sequence from the firstisolated polypeptide.

In one embodiment, the polypeptide includes at least two modificationsselected from the (a)-(d) modifications. In an exemplary embodiment, thepolypeptide includes (a) at least one crosslink between a side chain ofan aspartic acid residue of the polypeptide and a side chain of a lysineresidue of the polypeptide and (b) at least one crosslink between a sidechain of a glutamic acid residue of the polypeptide and a side chain ofa lysine residue of the polypeptide. In a further embodiment, thepolypeptide includes at least three modifications selected from the(a)-(d) modifications. In yet a further embodiment, the polypeptideincludes the (a), (b), (c), and (d) modifications.

When more than one mutant polypeptide is present during chemicalmodification by EDC, in one embodiment, the resulting compositionincludes at least one of any of the (a)-(h) modifications. In oneembodiment, the composition includes at least two modifications selectedfrom the (a)-(h) modifications. In a further embodiment, the compositionincludes at least three modifications selected from the (a)-(h)modifications. In yet a further embodiment, the composition includes atleast four modifications selected from the (a)-(h) modifications. Inanother embodiment, the composition includes at least one of each of the(a)-(h) modifications.

In an exemplary embodiment, the resulting composition includes (a) atleast one crosslink between a side chain of an aspartic acid residue ofthe polypeptide and a side chain of a lysine residue of the polypeptide;and (b) at least one crosslink between a side chain of a glutamic acidresidue of the polypeptide and a side chain of a lysine residue of thepolypeptide. In one embodiment, the composition further includes (c) atleast one crosslink between the carboxyl group at the C-terminus of thepolypeptide and the amino group of the N-terminus of the polypeptide;and (d) at least one crosslink between the carboxyl group at theC-terminus of the polypeptide and a side chain of a lysine residue ofthe polypeptide.

In another exemplary embodiment, the resulting composition includes (e)at least one crosslink between a side chain of an aspartic acid residueof the polypeptide and a side chain of a lysine residue of a secondisolated polypeptide; (f) at least one crosslink between a side chain ofa glutamic acid residue of the polypeptide and a side chain of a lysineresidue of a second isolated polypeptide; (g) at least one crosslinkbetween the carboxyl group at the C-terminus of the polypeptide and theamino group of the N-terminus of a second isolated polypeptide; and (h)at least one crosslink between the carboxyl group at the C-terminus ofthe polypeptide and a side chain of a lysine residue of a secondisolated polypeptide.

In a further exemplary embodiment, the resulting composition includes(a) at least one crosslink between a side chain of an aspartic acidresidue of the polypeptide and a side chain of a lysine residue of thepolypeptide; (b) at least one crosslink between a side chain of aglutamic acid residue of the polypeptide and a side chain of a lysineresidue of the polypeptide; (e) at least one crosslink between a sidechain of an aspartic acid residue of the polypeptide and a side chain ofa lysine residue of a second isolated polypeptide; and (f) at least onecrosslink between a side chain of a glutamic acid residue of thepolypeptide and a side chain of a lysine residue of a second isolatedpolypeptide.

In a preferred embodiment, the chemical crosslinking agent includesformaldehyde, more preferably, an agent including formaldehyde in theabsence of lysine. Glycine or other appropriate compound with a primaryamine can be used as the quencher in crosslinking reactions.Accordingly, in another preferred embodiment, the chemical agentincludes formaldehyde and use of glycine.

In yet another preferred embodiment, the chemical crosslinking agentincludes EDC and NHS. As is known in the art, NHS may be included in EDCcoupling protocols. However, the inventors surprisingly discovered thatNHS may facilitate in further decreasing cytotoxicity of the mutant C.difficile toxin, as compared to the corresponding wild-type toxin, ascompared to a genetically mutated toxin, and as compared to agenetically mutated toxin that has been chemically crosslinked by EDC.See, for example, Example 22. Accordingly, without being bound bymechanism or theory, a mutant toxin polypeptide having a beta-alaninemoiety linked to a side chain of at least one lysine residue of thepolypeptide (e.g., resulting from a reaction of the mutant toxinpolypeptide, EDC, and NHS) may facilitate in further decreasingcytotoxicity of the mutant toxin, as compared to, for example, a C.difficile toxin (wild-type or mutant) wherein a beta-alanine moiety isabsent.

Use of EDC and/or NHS may also include use of glycine or otherappropriate compound with a primary amine as the quencher. Any compoundhaving a primary amine may be used as a quencher, such as, for exampleglycine methyl ester and alanine. In a preferred embodiment, thequencher compound is a non-polymeric hydrophilic primary amine. Examplesof a non-polymeric hydrophilic primary amine include, for example, aminosugars, amino alcohols, and amino polyols. Specific examples of anon-polymeric hydrophilic primary amine include glycine, ethanolamine,glucamine, amine functionalized polyethylene glycol, and aminefunctionalized ethylene glycol oligomers.

In one aspect, the invention relates to a mutant C. difficile toxinpolypeptide having at least one amino acid side chain chemicallymodified by EDC and a non-polymeric hydrophilic primary amine,preferably glycine. The resulting glycine adducts (e.g., from a reactionof triple mutant toxins treated with EDC, NHS, and quenched withglycine) may facilitate in decreasing cytotoxicity of the mutant toxinas compared to the corresponding wild-type toxin.

In one embodiment, when a mutant C. difficile toxin polypeptide ischemically modified by EDC and glycine, the polypeptide includes atleast one modification when the polypeptide is modified by EDC (e.g., atleast one of any of the (a)-(h) modifications described above), and atleast one of the following exemplary modifications: (i) a glycine moietylinked to the carboxyl group at the C-terminus of the polypeptide; a) aglycine moiety linked to a side chain of at least one aspartic acidresidue of the polypeptide; and (k) a glycine moiety linked to a sidechain of at least one glutamic acid residue of the polypeptide. See, forexample, FIG. 24 and FIG. 25.

In one embodiment, at least one amino acid of the mutant C. difficileTcdA is chemically crosslinked and/or at least one amino acid of themutant C. difficile TcdB is chemically crosslinked. Any of the mutanttoxins described herein may be chemically crosslinked. In anotherembodiment, at least one amino acid of SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 7, and/or SEQ ID NO: 8 is chemically crosslinked. In oneembodiment, at least one amino acid residue of a polypeptide having theamino acid sequence of any of SEQ ID NOs: 1 through SEQ ID NO: 761 iscrosslinked. In another embodiment, at least one amino acid residue of apolypeptide having the amino acid sequence of any of SEQ ID NOs: 183through SEQ ID NO: 761 includes a modification as described above, e.g.,any of the (a)-(k) modifications, such as (a) at least one crosslinkbetween a side chain of an aspartic acid residue of the polypeptide anda side chain of a lysine residue of the polypeptide.

For example, the at least one amino acid may be chemically crosslinkedby an agent that includes a carbodiimide, such as EDC. Carbodiimides mayform a covalent bond between free carboxyl (e.g., from the side chainsof aspartic acid and/or glutamic acid) and amino groups (e.g., in theside chain of lysine residues) to form stable amide bonds.

As another example, the at least one amino acid may be chemicallycrosslinked by an agent that includes NHS. NHS ester-activatedcrosslinkers may react with primary amines (e.g., at the N-terminus ofeach polypeptide chain and/or in the side chain of lysine residues) toyield an amide bond.

In another embodiment, the at least one amino acid may be chemicallycrosslinked by an agent that includes EDC and NHS. For example, in oneembodiment, the invention relates to an isolated polypeptide having theamino acid sequence set forth in SEQ ID NO: 4, wherein the methionineresidue at position 1 is optionally not present, wherein the polypeptideincludes at least one amino acid side chain chemically modified by EDCand NHS. In another embodiment, the invention relates to an isolatedpolypeptide having the amino acid sequence set forth in SEQ ID NO: 6,wherein the methionine residue at position 1 is optionally not present,wherein the polypeptide includes at least one amino acid side chainchemically modified by EDC and NHS. In yet another embodiment, theinvention relates to an isolated polypeptide having the amino acidsequence set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 83, SEQID NO: 85, SEQ ID NO: 7, or SEQ ID NO: 8. The polypeptide is modified bycontacting the polypeptide with EDC and NHS. See, for example, FIG. 24and FIG. 25.

When a mutant C. difficile toxin polypeptide is chemically modified by(e.g., by contacting) EDC and NHS, in one embodiment, the polypeptideincludes at least one modification when the polypeptide is modified byEDC (e.g., at least one of any of the (a)-(h) modifications describedabove), and (l) a beta-alanine moiety linked to a side chain of at leastone lysine residue of the polypeptide.

In another aspect, the invention relates to a mutant C. difficile toxinpolypeptide wherein the polypeptide includes at least one amino acidside chain chemically modified by EDC, NHS, and a non-polymerichydrophilic primary amine, preferably glycine. In one embodiment, thepolypeptide includes at least one modification when the polypeptide ismodified by EDC (e.g., at least one of any of the (a)-(h) modificationsdescribed above), at least one modification when the polypeptide ismodified by glycine (e.g., at least one of any of the (i)-(k)modifications described above), and (l) a beta-alanine moiety linked toa side chain of at least one lysine residue of the polypeptide. See, forexample, FIG. 24 and FIG. 25.

In one aspect, the invention relates to a mutant C. difficile toxinpolypeptide, wherein a side chain of at least one lysine residue of thepolypeptide is linked to a beta-alanine moiety. In one embodiment, aside chain of a second lysine residue of the polypeptide is linked to aside chain of an aspartic acid residue and/or to a side chain of aglutamic acid residue. The “second” lysine residue of the polypeptideincludes a lysine residue of the polypeptide that is not linked to abeta-alanine moiety. The side chain of an aspartic acid and/or the sidechain of a glutamic acid to which the second lysine residue is linkedmay be that of the polypeptide to form an intra-molecular crosslink, orthat of a second polypeptide to form an inter-molecular crosslink. Inanother embodiment, a side chain of at least one aspartic acid residueand/or a side chain of at least one glutamic acid residue of thepolypeptide is linked to a glycine moiety. The aspartic acid residueand/or the glutamic acid residue that is linked to a glycine moiety isnot also linked to a lysine residue.

In another aspect, the invention relates to a mutant C. difficile toxinwherein at least one amino acid side chain of a wild-type C. difficiletoxin is chemically modified. In one embodiment, at least one amino acidside chain of a wild-type C. difficile toxin A and/or at least one aminoacid side chain of a wild-type C. difficile toxin B is chemicallymodified by EDC. For example, in one embodiment, TcdA (SEQ ID NO: 1)and/or Tcdb (SEQ ID NO: 2) is chemically modified by EDC. In anotherembodiment, the wild-type toxin is chemically modified by EDC and NHS.In one embodiment, the mutant toxin includes a chemically modifiedwild-type toxin A, wherein the wild-type toxin A is any one described inTable 1-a. In another embodiment, the mutant toxin includes a chemicallymodified wild-type toxin B, wherein the wild-type toxin B is any onedescribed in Table 2-a.

As yet another example of a chemically crosslinked mutant C. difficiletoxin polypeptide, the at least one amino acid may be chemicallycrosslinked by an agent that includes formaldehyde. Formaldehyde mayreact with the amino group of an N-terminal amino acid residue and theside-chains of arginine, cysteine, histidine, and lysine. Formaldehydeand glycine may form a Schiff-base adduct, which may attach to primaryN-terminal amino groups, arginine, and tyrosine residues, and to alesser degree asparagine, glutamine, histidine, and tryptophan residues.

A chemical crosslinking agent is said to reduce cytotoxicity of a toxinif the treated toxin has less toxicity (e.g., about 100%, 99%, 95%, 90%,80%, 75%, 60%, 50%, 25%, or 10% less toxicity) than untreated toxinunder identical conditions, as measured, for example, by an in vitrocytotoxicity assay, or by animal toxicity.

Preferably, the chemical crosslinking agent reduces cytotoxicity of themutant C. difficile toxin by at least about a 2-log₁₀ reduction, morepreferably about a 3-log₁₀ reduction, and most preferably about a4-log₁₀ or more, relative to the mutant toxin under identical conditionsbut in the absence of the chemical crosslinking agent. As compared tothe wild-type toxin, the chemical crosslinking agent preferably reducescytotoxicity of the mutant toxin by at least about a 5-log₁₀ reduction,about a 6-log₁₀ reduction, about a 7-log₁₀ reduction, about an 8-log₁₀reduction, or more.

In another preferred embodiment, the chemically inactivated mutant C.difficile toxin exhibits EC₅₀ value of greater than or at least about 50μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml,700 μg/ml, 800 μg/ml, 900 μg/ml, 1000 μg/ml or greater, as measured by,for example, an in vitro cytotoxicity assay, such as one describedherein.

Reaction conditions for contacting the mutant toxin with the chemicalcrosslinking agent are within the scope of expertise of one skilled inthe art, and the conditions may vary depending on the agent used.However, the inventors surprisingly discovered optimal reactionconditions for contacting a mutant C. difficile toxin polypeptide with achemical crosslinking agent, while retaining functional epitopes anddecreasing cytotoxicity of the mutant toxin, as compared to thecorresponding wild-type toxin.

Preferably, the reaction conditions are selected for contacting a mutanttoxin with the crosslinking agent, wherein the mutant toxin has aminimum concentration of about 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0mg/ml to a maximum of about 3.0, 2.5, 2.0, 1.5, or 1.25 mg/ml. Anyminimum value may be combined with any maximum value to define a rangeof suitable concentrations of a mutant toxin for the reaction. Mostpreferably, the mutant toxin has a concentration of about 1.0-1.25 mg/mlfor the reaction.

In one embodiment, the agent used in the reaction has a minimumconcentration of about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, 20mM, 30 mM, 40 mM, or 50 mM, and a maximum concentration of about 100 mM,90 mM, 80 mM, 70 mM, 60 mM, or 50 mM. Any minimum value may be combinedwith any maximum value to define a range of suitable concentrations ofthe chemical agent for the reaction.

In a preferred embodiment wherein the agent includes formaldehyde, theconcentration used is preferably any concentration between about 2 mM to80 mM, most preferably about 40 mM. In another preferred embodimentwherein the agent includes EDC, the concentration used is preferably anyconcentration between about 1.3 mM to about 13 mM, more preferably about2 mM to 3 mM, most preferably about 2.6 mM. In one embodiment, theconcentration of EDC is at most 5 g/L, 4 g/L, 3 g/L, 2.5 g/L, 2 g/L, 1.5g/L, 1.0 g/L, 0.5 g/L based on the total reaction volume, preferably atmost 1 g/L, more preferably at most 0.5 g/L.

Exemplary reaction times in which the mutant toxin is contacted with thechemical crosslinking agent include a minimum of about 0.5, 1, 2, 3, 4,5, 6, 12, 24, 36, 48, or 60 hours, and a maximum of about 14 days, 12days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day, or 12 hours, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour. Any minimum value may be combinedwith any maximum value to define a range of suitable reaction times.

In a preferred embodiment, the step of contacting the mutant toxin withthe chemical crosslinking agent occurs for a period of time that issufficient to reduce cytotoxicity of the mutant C. difficile toxin to anEC₅₀ value of at least about 1000 μg/ml in a suitable human cell, e.g.,IMR-90 cells, in a standard in vitro cytotoxicity assay, as compared toan identical mutant toxin in the absence of the crosslinking agent. Morepreferably, the reaction step is carried out for a time that is at leasttwice as long, and most preferably at least three times as long or more,as the period of time sufficient to reduce the cytotoxicity of themutant toxin to an EC₅₀ value of at least about 1000 μg/ml in a suitablehuman cell. In one embodiment, the reaction time does not exceed about168 hours (or 7 days).

For example, in one embodiment wherein the agent includes formaldehyde,the mutant toxin is preferably contacted with the agent for about 12hours, which was shown to be an exemplary period of time that wassufficient to reduce cytotoxicity of the mutant C. difficile toxin to anEC₅₀ value of at least about 1000 μg/ml in a suitable human cell, e.g.,IMR-90 cells, in a standard in vitro cytotoxicity assay, as compared toan identical mutant toxin in the absence of the crosslinking agent. In amore preferred embodiment, the reaction is carried out for about 48hours, which is at least about three times as long as a sufficientperiod of time for the reaction. In such an embodiment, the reactiontime is preferably not greater than about 72 hours.

In another embodiment wherein the agent includes EDC, the mutant toxinis preferably contacted with the agent for about 0.5 hours, morepreferably at least about 1 hour, or most preferably about 2 hours. Inone embodiment, the mutant toxin is contacted with EDC for at most about5 hours, preferably at most about 3 hours, more preferably at most about2 hours. In such an embodiment, the reaction time is preferably notgreater than about 6 hours.

Exemplary pH at which the mutant toxin is contacted with the chemicalcrosslinking agent include a minimum of about pH 5.5, 6.0, 6.5, 7.0, or7.5, and a maximum of about pH 8.5, 8.0, 7.5, 7.0, or 6.5. Any minimumvalue may be combined with any maximum value to define a range ofsuitable pH. Preferably, the reaction occurs at pH 6.5 to 7.5,preferably at pH 7.0.

Exemplary temperatures at which the mutant toxin is contacted with thechemical crosslinking agent include a minimum of about 2° C., 4° C., 10°C., 20° C., 25° C., or 37° C., and a maximum temperature of about 40°C., 37° C., 30° C., 27° C., 25° C., or 20° C. Any minimum value may becombined with any maximum value to define a range of suitable reactiontemperature. Preferably, the reaction occurs at about 20° C. to 30° C.,most preferably at about 25° C.

The immunogenic compositions described above may include one mutant C.difficile toxin (A or B). Accordingly, the immunogenic compositions canoccupy separate vials (e.g., a separate vial for a composition includingmutant C. difficile toxin A and a separate vial for a compositionincluding mutant C. difficile toxin B) in the preparation or kit. Theimmunogenic compositions may be intended for simultaneous, sequential,or separate use.

In another embodiment, the immunogenic compositions described above mayinclude both mutant C. difficile toxins (A and B). Any combination ofmutant C. difficile toxin A and mutant C. difficile toxin B describedmay be combined for an immunogenic composition. Accordingly, theimmunogenic compositions can be combined in a single vial (e.g., asingle vial containing both a composition including mutant C. difficileTcdA and a composition including mutant C. difficile TcdB). Preferably,the immunogenic compositions include a mutant C. difficile TcdA and amutant C. difficile TcdB.

For example, in one embodiment, the immunogenic composition includes SEQID NO: 4 and SEQ ID NO: 6, wherein at least one amino acid of each ofSEQ ID NO: 4 and SEQ ID NO: 6 is chemically crosslinked. In anotherembodiment, the immunogenic composition includes a mutant C. difficiletoxin A, which includes SEQ ID NO: 4 or SEQ ID NO: 7, and a mutant C.difficile toxin B, which comprises SEQ ID NO: 6 or SEQ ID NO: 8, whereinat least one amino acid of each of the mutant C. difficile toxins ischemically crosslinked.

In another embodiment, the immunogenic composition includes any sequenceselected from SEQ ID NO: 4, SEQ ID NO: 84, and SEQ ID NO: 83, and anysequence selected from SEQ ID NO: 6, SEQ ID NO: 86, and SEQ ID NO: 85.In another embodiment, the immunogenic composition includes SEQ ID NO:84 and an immunogenic composition including SEQ ID NO: 86. In anotherembodiment, the immunogenic composition includes SEQ ID NO: 83 and animmunogenic composition including SEQ ID NO: 85. In another embodiment,the immunogenic composition includes SEQ ID NO: 84, SEQ ID NO: 83, SEQID NO: 86, and SEQ ID NO: 85.

In another embodiment, the immunogenic composition includes apolypeptide having any one sequence selected from SEQ ID NO: 1 to SEQ IDNO: 761, and a second polypeptide having any one sequence selected fromSEQ ID NO: 1 to SEQ ID NO: 761.

It is understood that any of the inventive compositions, for example,immunogenic compositions including a mutant toxin A and/or mutant toxinB, can be combined in different ratios or amounts for therapeuticeffect. For example, the mutant C. difficile TcdA and mutant C.difficile TcdB can be present in a immunogenic composition at a ratio inthe range of 0.1:10 to 10:0.1, A:B. In another embodiment, for example,the mutant C. difficile TcdB and mutant C. difficile TcdA can be presentin a immunogenic composition at a ratio in the range of 0.1:10 to10:0.1, B:A. In one preferred embodiment, the ratio is such that thecomposition includes a greater total amount of a mutant TcdB than atotal amount of mutant TcdA.

In one aspect, an immunogenic composition is capable of binding to aneutralizing antibody or binding fragment thereof. Preferably, theneutralizing antibody or binding fragment thereof is one describedherein below. In one exemplary embodiment, an immunogenic composition iscapable of binding to an anti-toxin A antibody or binding fragmentthereof, wherein the anti-toxin A antibody or binding fragment thereofincludes a variable light chain having the amino acid sequence of SEQ IDNO: 36 and a variable heavy chain having the amino acid sequence of SEQID NO: 37. For example, the immunogenic composition may include a mutantC. difficile TcdA, SEQ ID NO: 4, or SEQ ID NO: 7. As another example,the immunogenic composition may include SEQ ID NO: 84 or SEQ ID NO: 83.

In another exemplary embodiment, an immunogenic composition is capableof binding to an anti-toxin B antibody or binding fragment thereof,wherein the anti-toxin B antibody or binding fragment thereof includes avariable light chain of B8-26 and a variable heavy chain of B8-26. Forexample, the immunogenic composition may include a mutant C. difficileTcdB, SEQ ID NO: 6, or SEQ ID NO: 8. As another example, the immunogeniccomposition may include SEQ ID NO: 86 or SEQ ID NO: 85.

Recombinant Cell

In another aspect, the invention relates to a recombinant cell orprogeny thereof. In one embodiment, the cell or progeny thereof includesa polynucleotide encoding a mutant C. difficile TcdA and/or a mutant C.difficile TcdB.

In another embodiment, the recombinant cell or progeny thereof includesa nucleic acid sequence that encodes a polypeptide having at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, morepreferably about 99% or most preferably about 100% identity to any ofSEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights. In one embodiment, the recombinant cell or progeny thereofincludes a nucleic acid sequence that encodes a polypeptide having atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about98%, more preferably about 99% or most preferably about 100% identity toany of SEQ ID NOs: 1 through SEQ ID NO: 761.

In another embodiment, the recombinant cell or progeny thereof includesa nucleic acid sequence that encodes a polypeptide having at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, morepreferably about 99% or most preferably about 100% identity to any ofSEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 83, or SEQ ID NO: 85, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights.

In an additional embodiment, the recombinant cell or progeny thereofincludes nucleic acid sequence having at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% ormost preferably about 100% identity to any of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 45, SEQ IDNO: 46, or SEQ ID NO: 47, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights. The recombinant cellmay be derived from any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.Preferably, the recombinant cell is derived from any cell that issuitable for expressing heterologous nucleic acid sequences greater thanabout 5000, 6000, preferably about 7000, and more preferably about 8000nucleotides or more. The prokaryotic host cell may be any gram-negativeor gram-positive bacterium. In exemplary embodiments, the prokaryotichost cell lacks an endogenous polynucleotide encoding a toxin and/orspore.

Gram-negative bacteria include, but are not limited to, Campylobacter,E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, and Ureaplasma. For example, therecombinant cell may be derived from a Pseudomonas fluorescens cell, asdescribed in US patent application publication 2010013762, paragraphs[0201]-[0230], which is incorporated herein by reference.

Gram-positive bacteria include, but are not limited to, Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.Preferably, the cell is derived from a C. difficile cell.

The inventors identified strains of wild-type C. difficile that lack anendogenous polynucleotide encoding a C. difficile toxin. The strainslacking endogenous toxin A and B genes include the following strains,which are available through the American Type Culture Collection (ATCC)(Manassas, Va.): C. difficile 1351 (ATCC 43593™) C. difficile 3232 (ATCCBAA-1801™), C. difficile 7322 (ATCC 43601™), C. difficile 5036 (ATCC43603™), C. difficile 4811 (ATCC 43602™), and C. difficile VPI 11186(ATCC 700057™)

Accordingly, in one embodiment, the recombinant C. difficile cell isderived from a strain described herein. Preferably, the recombinant C.difficile cell or progeny thereof is derived from the group consistingof C. difficile 1351, C. difficile 5036, and C. difficile VPI 11186.More preferably, the recombinant C. difficile cell or progeny thereof isderived from a C. difficile VPI 11186 cell.

In a preferred embodiment, the sporulation gene of the recombinant C.difficile cell or progeny thereof is inactivated. Spores may beinfective, highly resistant, and facilitate the persistence of C.difficile in aerobic environments outside of the host. Spores may alsocontribute to survival of C. difficile inside the host duringantimicrobial therapy. Accordingly, a C. difficile cell lacking asporulation gene is useful to produce a safe immunogenic composition foradministration to mammals. In addition, use of such cells facilitatessafety during manufacturing, e.g., safety to protect the facility,future products, and staff.

Examples of sporulation genes for targeted inactivation include, interalia, spo0A, spollE, σ^(E), σ^(G), and σ^(k). Preferably, the spo0A geneis inactivated.

Methods of inactivating a C. difficile sporulation gene are known in theart. For example, a sporulation gene may be inactivated by targetedinsertion of a selectable marker, such as, an antibiotic resistancemarker. See, for example, Heap et al., J Microbiol Methods. 2010January; 80(1):49-55; Heap et al., J. Microbiol. Methods, 2007September; 70(3):452-464; and Underwood et al., J Bacteriol. 2009December; 191(23):7296-305. See also, for example, Minton et al.,WO2007/148091, entitled, “DNA Molecules and Methods,” incorporatedherein by reference in its entirety from pages 33-66, or thecorresponding US publication US 20110124109 A1, paragraphs[00137]-[0227].

Compositions Relating to Culturing a C. difficile Cell

The invention further relates to compositions and methods for use inculturing C. difficile and producing C. difficile toxins. In one aspect,the invention relates to a culture medium comprising a nitrogen sourceand a C. difficile cell.

Suitable culture medium nitrogen sources include: HY-SOY (Quest),AMI-SOY (Quest), NZ-SOY (Quest), NZ-SOY BL4 (Quest), NZ-SOY BL7 (Quest),SHEFTONE D (Sheffield), SE50M (DMV), SE50 (DMV), SE %) MK (DMV), SOYPEPTONE (Gibco), BACTO-SOYTON (Difco), NUTRISOY 2207 (ADM), BAKESNUTRISOY (ADM) NUTRISOY FLOUR, Soybean meal, BACTO-YEAST EXTRACT (Difco)YEAST EXTRACT (Gibco), HY-YEST 412 yeast extract (Quest), HY-YEST 441yeast extract (Quest), HY-YEST 444 yeast extract (Quest), HY-YEST 455yeast extract (Quest) BACTO-MALT EXTRACT (Difco), Corn Steep, and PROFLO(Traders).

In one aspect, the invention relates to a culture medium including avegetable hydrolysate and a C. difficile cell. Any vegetable hydrolysatemay be suitable. Examples of suitable vegetable hydrolysates arecottonseed hydrolysate, pea hydrolysate, and soy hydrolysate.

In a preferred embodiment, the vegetable hydrolysate is soy hydrolysate.Preferably, the soy hydrolysate is SE50MK (Friesland-Campaigna). Otherexamples of soy products that may be used in the invention, and theirsources, include: Tekniscience: Soy Peptone A1, Soy Peptone A2, SoyPeptone A3, Plant Peptone E1, Plant Peptone ET1, and Wheat Peptone E1;Quest: HY-Soy, HY-Soy T, AMI-Soy, NZ-Soy, NZ-Soy BLA, and NZ-Soy BL7;DMV: SE50M, SE70M, SE50MK, SE50MK-NK (Friesland-Campaigna), WGE80BT,WGE80M, CNE50M, and SE70BT; Marcor: Soy Peptone Type AB, Soy PeptoneType AC, Soy Peptone Type SL, Soy Peptone Type II, and Soy Peptone TypeF; Oxoid: Vegetable Peptone and Vegetable Peptone No. 1; Gibco: SoyPeptone; and Difco: Bacsoytone.

Concentrations of the vegetable hydrolysate in the culture medium canrange, for example, between a minimal value of 5, 10, 20, 30, 40, or 50g/L to a maximal value of 200, 150, 100, or 75 g/L. Any minimum valuecan be combined with any maximum value to define a suitable range. In apreferred embodiment, the concentration of vegetable hydrolysate in theculture medium is between 10-50 g/L, most preferably about 30 g/L. Theconcentration of vegetable hydrolysate in the culture medium describedherein may be based on the total volume of the culture medium.

In another aspect, the invention relates to a culture medium including ayeast extract (e.g., as a nitrogen source) and a Clostridium difficilecell. Most preferably, the yeast extract is HY YEST 412 (KerryBiosciences).

Concentrations of the yeast extract in the culture medium can range, forexample, between a minimal value of 5, 10, 20, 30, 40, or 50 g/L to amaximal value of 200, 150, 100, or 75 g/L. Any minimum value can becombined with any maximum value to define a suitable range. In apreferred embodiment, the concentration of yeast extract in the culturemedium is between 10-50 g/L, most preferably about 20 g/L. Theconcentration of yeast extract in the culture medium described hereinmay be based on the total volume of the culture medium.

The inventors discovered that growth of C. difficile can be supported ina culture medium including a vegetable hydrolysate without yeastextract, and in a culture medium that includes yeast extract without avegetable hydrolysate. The culture of cells and/or production of toxinresulting from a culture medium that includes both a vegetablehydrosylate and yeast extract, however, has been observed to be higherthan the yields resulting from a culture medium including a vegetablehydrolysate in the absence of yeast extract, and higher than the yieldsresulting from a culture medium including yeast extract in the absenceof a vegetable hydrolysate.

Accordingly, the inventors discovered that a combination of a vegetablehydrolysate and yeast extract helps to support maximal growth of C.difficile and/or production of toxin. In one aspect, the inventionrelates to a culture medium including a vegetable hydrolysate, yeastextract, and a C. difficile cell. The vegetable hydrolysate can be anysuitable vegetable hydrolysate known in the art as described above.Preferably, the hydrolysate is soy hydrolysate. More preferably, the soyhydrolysate is SE50MK (Friesland-Campaigna). In a preferred embodiment,the yeast extract is HY YEST 412.

In one embodiment, the medium does not include a carbon source. Theinventors observed that culture medium including soy hydrolysate/yeastextract in the absence of a carbon source achieves OD₆₀₀ values of 2-3and toxin production yields of 10-15 mg/L.

However, a culture medium that includes a carbon source was surprisinglyobserved to increase the culture of C. difficile cells and production oftoxin, as compared to a medium in the absence of a carbon source. Inaddition, the inventors surprisingly discovered that production of themutant toxin was not markedly inhibited by culturing the C. difficilecell under anaerobic conditions including a carbon source in thepresence of a medium including soy hydrolysate. Accordingly, in apreferred embodiment, the medium further includes a carbon source. Morepreferably, in one embodiment, the culture medium includes a carbonsource, wherein the medium further includes a recombinant C. difficilecell. Even more preferably, the recombinant C. difficile cell includes aconstitutive promoter. In a preferred embodiment, the promoter is aClostridium sporogenes feredoxin (fdx) promoter. In another preferredembodiment, the recombinant C. difficile cell does not include aregulated chromosomal promoter that may be negatively regulated by, forexample, glucose repression. In a most preferred embodiment, the C.difficile cell is a recombinant cell or progeny thereof as describedabove. Any carbon source may be used in the culture medium. Suitablecarbon sources include glucose, dextrose, mannitol, fructose, and/ormannose. Preferably, the carbon source in the culture medium is glucose.In a preferred embodiment, the culture medium does not includearabinose, xylose, sucrose, lactose, maltose, glycerol, rhamnose, and/orgalactose.

Concentrations of a carbon source (e.g., glucose, mannitol, fructose,and/or mannose) in the culture medium can range, for example, between aminimal value of 1, 5, 10, 15, 20, 30, 40, 50, or 60 g/L to a maximalvalue of 150, 100, 90, 80, 75, 70, 50, 40, or 30 g/L. Any minimum valuecan be combined with any maximum value to define a suitable range. In apreferred embodiment, the concentration of a carbon source in theculture medium is between 5-70 g/L. In one embodiment, the concentrationof a carbon source in the culture medium is about 10 g/L. In anotherembodiment, the concentration of a carbon source in the culture mediumis greater than 10 g/L. In another embodiment, the concentration of acarbon source (e.g., glucose) in the culture medium is 45-75 g/L,preferably 55-65 g/L more preferably 55-65 g/L. In another embodiment,the concentration of a carbon source in the culture medium is about 60g/L. The concentration of carbon source in the culture medium describedherein may be based on the total volume of the culture medium.

In one embodiment, the culture medium further includes a chloramphenicolderivative. Exemplary chloramphenicol derivatives include any one ofthiamphenicol, florfenicol, chloramphenicol succinate, andfluoramphenicol. Preferably, the culture medium includes thiamphenicol.Without being bound by mechanism or theory, the chloramphenicolderivative is believed to help in preventing plasmid loss duringfermentation and in increasing production of cells and/or toxin, ascompared to a culture medium in the absence of a chloramphenicolderivative.

Concentrations of a chloramphenicol derivative in the culture medium canrange, for example, between a minimal value of 5, 10, 15, 20, or 30 mg/Lto a maximal value of 100, 75, 50, or 40 mg/L. In another embodiment,the concentration of chloramphenicol derivative in the culture mediumcan range, for example between a minimal value of 0.5 g/L, 1 g/L, 1.5g/L, 2 g/L, 2.5 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, or 5 g/L to amaximal value of 10 g/L, 9.5 g/L, 9 g/L, 8.5 g/L, 8 g/L, 7.5 g/L, 7 g/L,6.5 g/L, 6 g/L, 5.5 g/L, 5 g/L. Any minimum value can be combined withany maximum value to define a suitable range. In a preferred embodiment,the concentration of a chloramphenicol derivative in the culture mediumis between 5-20 mg/L, most preferably about 15 mg/L. In a anotherpreferred embodiment, the concentration of a chloramphenicol derivativein the culture medium is between 1 g/L-10 g/L, preferably between 1g/L-5 g/L, most preferably about 3 g/L. The concentration ofchloramphenicol derivative in the culture medium described herein may bebased on the total volume of the culture medium.

In one embodiment, the culture medium further includes a cellprotectant, such as polyethylene glycol, a polyvinyl alcohol or apluronic polyol. In one embodiment, the culture medium includespolyethylene glycol, such as polyethylene glycol 2000 (PPG 2000).

Concentrations of a cell protectant, such as polyethylene glycol, in theculture medium can range, for example, between a minimal value of 0.01,0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60,0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 ml/L to a maximal valueof 2, 1, 0.90, 0.80, 0.70, 0.60, 0.50, 0.40, 0.30, 0.20 ml/L. Anyminimum value can be combined with any maximum value to define asuitable range. In a preferred embodiment, the concentration of a thecell protectant, such as polyethylene glycol in the culture medium isbetween 0.01 ml/L to 0.0.50, most preferably about 0.25 ml/L. Theconcentration of cell protectant in the culture medium described hereinmay be based on the total volume of the culture medium.

In a preferred embodiment, the culture medium includes a vegetablehydrolysate, yeast extract, and a carbon source, wherein the vegetablehydrolysate is soy hydrolysate, wherein the yeast extract is HY YEST 412(Kerry Biosciences), and wherein the carbon source is glucose, mannitol,fructose, and/or mannose. In a most preferred embodiment, the culturemedium includes soy hydrolysate SE50MK, HY YEST 412 yeast extract,glucose, and thiamphenicol, at pH 7.

In one preferred embodiment, the culture medium includes 30 g/L soyhydrolysate SE50MK, 20 g/L HY YEST 412 yeast extract, 10 g/L glucose,and 15 mg/L thiamphenicol, at pH 7. In one preferred embodiment, theculture medium includes about 30 g/L soy hydrolysate SE50MK, about 20g/L HY YEST 412 yeast extract, about 10 g/L glucose, and about 15 mg/Lthiamphenicol, at pH 7. The culture medium may further include aboutabout 0.25 ml/L PPG 2000. In another embodiment, the culture medium mayfurther include dextrose.

In another preferred embodiment, the culture medium includes 30 g/L soyhydrolysate SE50MK, 20 g/L HY YEST 412 yeast extract, 60 g/L glucose,and 15 mg/L thiamphenicol, at pH 7. In another preferred embodiment, theculture medium includes about 30 g/L soy hydrolysate SE50MK, about 20g/L HY YEST 412 yeast extract, about 60 g/L glucose, and about 15 mg/Lthiamphenicol, at pH 7. The culture medium may further include aboutabout 0.25 ml/L PPG 2000. In another embodiment, the culture medium mayfurther include dextrose.

In one embodiment, the culture medium further includes polypropyleneglycol 2000 (PPG 2000). The culture medium may include about 0.05 mL PPG2000 to about 1 ml PPG 2000/L medium, preferably the culture mediumincludes about 0.25 ml/L PPG 2000/L medium.

The culture medium described herein may be suitable for culturing any C.difficile cell. In one embodiment, the cell is not genetically modified.In another embodiment, the cell is genetically modified, such as arecombinant cell or progreny thereof as described above. In oneembodiment, the C. difficile cell is lacks an endogenous polynucleotideencoding a toxin. In a preferred embodiment, the C. difficile cell isderived from VPI 11186.

In another embodiment, the cell includes a constitutive promoter. In apreferred embodiment, the promoter is a Clostridium sporogenes feredoxin(fdx) promoter. In another preferred embodiment, the cell does notinclude a regulated chromosomal promoter that may be negativelyregulated by, for example, glucose repression. In a most preferredembodiment, the C. difficile cell is a recombinant cell or progenythereof as described above.

The inventors also discovered that a monoclonal antibody medium supportsgrowth of C. difficile. Accordingly, in one aspect, the inventionrelates to a culture medium that includes a monoclonal antibody medium.In one embodiment, the medium is SFM4MAb™ medium (Thermo Scientific).The medium was shown to give OD₆₀₀ values of about 10 and toxinproduction yield was about 40 mg/L.

In one embodiment, the pH of the culture medium can range, for example,between a minimal value of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, or 7.0 to a maximal value of 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4,7.3, 7.2, or 7.1. Any minimum value can be combined with any maximumvalue to define a suitable range. In a preferred embodiment, the pH ofthe culture medium is between 6.0 to 8.0, more preferably, between 6.5to 7.5. Most preferably, the pH is 7.0.

Suitable pH titrants are known in the art, including, for example,NH₄OH, Na₂CO₃, and NaOH. Preferably, the pH titrant is NaOH.

The culture medium may include phosphate-containing ingredients such asNa₂HPO₄, KH₂PO₄. However, in a preferred embodiment, the culture mediumdoes not include a phosphate-containing ingredient.

Methods Relating to Culturing a C. difficile Cell

In another aspect, the invention relates to a method of culturing C.difficile. The method includes culturing a C. difficile cell in a mediumas described above.

In one embodiment, growth of C. difficile according to the methods ofthe invention proceeds in at least two phases: seed growth andfermentation. A seed culture is first grown by inoculation from a stockculture, e.g., a working cell bank. The seed is used either to inoculatea second seed culture or to inoculate a relatively large fermentationculture. As is understood in the art, the number of seed cultures usedmay depend, for example, on the size and volume of the fermentationstep.

Accordingly, in one aspect, the invention relates to a method ofculturing C. difficile. The method includes culturing a C. difficilecell in a first culture medium under conditions that facilitate growthof the cell; inoculating a second culture medium with all or a portionof said first medium after said first culturing; culturing saidinoculated second medium under conditions that facilitate growth of thecell. The method may further include isolating a C. difficile toxin fromsaid second medium. In one embodiment, the C. difficile is grown in afirst culture medium referred to as a seed culture. In one embodiment,the seed culture includes a culture medium as described above and aninoculation from a stock culture that was grown in the medium.

The seed growth phase (or phases) are generally carried out to scale-upthe quantity of the microorganism from a stored culture, so that it canbe used as an inoculant for the fermentation phase. The seed growthphase can also be carried out to allow relatively dormant microbes instored cultures to become rejuvenated and to grow into actively growingcultures.

The volume and quantity of viable cells used to inoculate thefermentation culture can be controlled more accurately if taken from anactively growing culture (e.g., a seed culture), rather than if takenfrom a stored culture.

In addition, more than one (e.g., two or three) seed growth phases canbe used to scale-up the quantity of C. difficile for inoculation of thefermentation medium. Alternatively, growth of C. difficile in thefermentation phase can proceed directly from the stored culture bydirect inoculation, if desired.

The first culture medium includes a culture medium as described above.For example, in one embodiment, the first culture medium includes avegetable hydrolysate and a C. difficile cell. Preferably, the vegetablehydrolysate is soy hydrolysate, most preferably soy hydrolysate SE50MK.In another embodiment, the first culture medium includes a yeast extractand a C. difficile cell. Preferably, the yeast extract is HY YEST 412.In a further embodiment, the first culture medium includes a vegetablehydrolysate and a yeast extract. In yet a further embodiment, the firstculture medium further includes a carbon source. Preferably, the carbonsource is glucose. In one embodiment, the culture medium includes arecombinant C. difficile cell when a carbon source is included, whereinthe recombinant cell includes a constitutive promoter. In anotherembodiment, the first culture medium further includes a chloramphenicolderivative. Preferably, the culture medium includes thiamphenicol.

Concentrations of the vegetable hydrolysate in the first culture medium(e.g., seed culture medium) can range, for example, between a minimalvalue of 5, 10, 20, 30, 40, or 50 g/L to a maximal value of 200, 150,100, or 75 g/L. Any minimum value can be combined with any maximum valueto define a suitable range. In a preferred embodiment, the concentrationof vegetable hydrolysate in the culture medium is between 10-50 g/L,most preferably about 30 g/L. The concentration of vegetable hydrolysatein the culture medium described herein may be based on the total volumeof the culture medium.

Concentrations of the yeast extract in the first culture medium (e.g.,seed culture medium) can range, for example, between a minimal value of5, 10, 20, 30, 40, or 50 g/L to a maximal value of 200, 150, 100, or 75g/L. Any minimum value can be combined with any maximum value to definea suitable range. In a preferred embodiment, the concentration of yeastextract in the culture medium is between 10-50 g/L, most preferablyabout 20 g/L. The concentration of yeast extract in the culture mediumdescribed herein may be based on the total volume of the culture medium.

Concentrations of a carbon source in the first culture medium (e.g.,seed culture medium) can range, for example, between a minimal value of1, 5, 10, 15, or 20 g/L to a maximal value of 100, 75, 50, 40, or 30g/L. Any minimum value can be combined with any maximum value to definea suitable range. In a preferred embodiment, the concentration of acarbon source in the culture medium is between 5-20 g/L, most preferablyabout 10 g/L.

In one embodiment, the culture medium further includes a chloramphenicolderivative selected from the group consisting of thiamphenicol,florfenicol, chloramphenicol succinate, and fluoramphenicol. Preferably,the culture medium includes thiamphenicol. Concentrations of achloramphenicol derivative in the first culture medium (e.g., seedculture medium) can range, for example, between a minimal value of 5,10, 15, 20, or 30 mg/L to a maximal value of 100, 75, 50, or 40 mg/L.Any minimum value can be combined with any maximum value to define asuitable range. In a preferred embodiment, the concentration of achloramphenicol derivative in the culture medium is between 5-20 mg/L,most preferably about 15 mg/L.

In one embodiment, the pH of the first culture medium can range, forexample, between a minimal value of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, or 7.0 to a maximal value of 8.0, 7.9, 7.8, 7.7, 7.6,7.5, 7.4, 7.3, 7.2, or 7.1. Any minimum value can be combined with anymaximum value to define a suitable range. In a preferred embodiment, thepH of the first culture medium is between 6.0 to 8.0, more preferably,between 6.5 to 7.5. Most preferably, the pH is 7.0.

In a preferred embodiment, the first culture medium includes soyhydrolysate, yeast extract HY YEST 412, glucose, thiamphenicol, at pH 7.More preferably, the culture medium includes 30 g/L soy hydrolysateSE50MK, 20 g/L yeast extract HY YEST 412, 10 g/L glucose, 15 mg/Lthiamphenicol, at pH 7.

To start the fermentation phase, a portion or all of a seed culturecontaining C. difficile may be used to inoculate fermentation culturemedium. An appropriate concentration of seed culture to use to inoculatefermentation media can be determined by those of skill in this art andcan range, for example, from 0.1-10%. As specific examples,concentrations of 0.5, 1, 5, 5.5, 6, 6.25, 6.5, 7, 8, 9, 10% can beused.

Fermentation may be used to produce the maximum amount of cells in alarge-scale anaerobic environment. In one embodiment, the C. difficileis grown as a fermentation culture. In one embodiment, the fermentationculture was inoculated from a seed culture that was grown in the medium.

The second culture medium includes a culture medium as described above.For example, in one embodiment, the second culture medium includes avegetable hydrolysate and a C. difficile cell. Preferably, the vegetablehydrolysate is soy hydrolysate, most preferably soy hydrolysate SE50MK.In another embodiment, the second culture medium includes a yeastextract and a C. difficile cell. Preferably, the yeast extract is HYYEST 412. In a further embodiment, the second culture medium includes avegetable hydrolysate and a yeast extract. In yet a further embodiment,the second culture medium further includes a carbon source. Preferably,the carbon source is glucose. In one embodiment, the culture mediumincludes a recombinant C. difficile cell when a carbon source isincluded, wherein the recombinant cell includes a constitutive promoter.In another embodiment, the second culture medium further includes achloramphenicol derivative. Preferably, the culture medium includesthiamphenicol.

Concentrations of the vegetable hydrolysate in the second culture mediumcan range, for example, between a minimal value of 5, 10, 20, 30, 40, or50 g/L to a maximal value of 200, 150, 100, or 75 g/L. Any minimum valuecan be combined with any maximum value to define a suitable range. In apreferred embodiment, the concentration of vegetable hydrolysate in theculture medium is between 10-50 g/L, most preferably about 30 g/L. Theconcentration of vegetable hydrolysate in the culture medium describedherein may be based on the total volume of the culture medium.

Concentrations of the yeast extract in the second culture medium canrange, for example, between a minimal value of 5, 10, 20, 30, 40, or 50g/L to a maximal value of 200, 150, 100, or 75 g/L. Any minimum valuecan be combined with any maximum value to define a suitable range. In apreferred embodiment, the concentration of yeast extract in the culturemedium is between 10-50 g/L, most preferably about 20 g/L.

Concentrations of a carbon source in the second culture medium canrange, for example, between a minimal value of 10, 20, 30, 40, 50, or 60g/L to a maximal value of 150, 100, 90, 80, or 70 g/L. Any minimum valuecan be combined with any maximum value to define a suitable range. In apreferred embodiment, the concentration of a carbon source in theculture medium is between 50-70 g/L, most preferably about 60 g/L. Inone embodiment, the concentration of a carbon source in the secondculture medium is greater than the concentration of the carbon source inthe first culture medium. The concentration of carbon source in theculture medium described herein may be based on the total volume of theculture medium.

In one embodiment, the culture medium further includes a chloramphenicolderivative selected from the group consisting of thiamphenicol,florfenicol, chloramphenicol succinate, and fluoramphenicol. Preferably,the culture medium includes thiamphenicol. Concentrations of achloramphenicol derivative in the second culture medium can range, forexample, between a minimal value of 5, 10, 15, 20, or 30 mg/L to amaximal value of 100, 75, 50, or 40 mg/L. In another embodiment, theconcentration of chloramphenicol derivative in the culture medium canrange, for example between a minimal value of 0.5 g/L, 1 g/L, 1.5 g/L, 2g/L, 2.5 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, or 5 g/L to a maximalvalue of 10 g/L, 9.5 g/L, 9 g/L, 8.5 g/L, 8 g/L, 7.5 g/L, 7 g/L, 6.5g/L, 6 g/L, 5.5 g/L, 5 g/L. Any minimum value can be combined with anymaximum value to define a suitable range. In a preferred embodiment, theconcentration of a chloramphenicol derivative in the culture medium isbetween 5-20 mg/L, most preferably about 15 mg/L. In a another preferredembodiment, the concentration of a chloramphenicol derivative in theculture medium is between 1 g/L-10 g/L, preferably between 1 g/L-5 g/L,most preferably about 3 g/L. The concentration of chloramphenicolderivative in the culture medium described herein may be based on thetotal volume of the culture medium.

In one embodiment, the culture medium further includes a cellprotectant, such as polyethylene glycol, a polyvinyl alcohol or apluronic polyol. In one embodiment, the culture medium includespolyethylene glycol, such as polyethylene glycol 2000 (PPG 2000).

Concentrations of a cell protectant, such as polyethylene glycol, in theculture medium can range, for example, between a minimal value of 0.01,0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60,0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 ml/L to a maximal valueof 2, 1, 0.90, 0.80, 0.70, 0.60, 0.50, 0.40, 0.30, 0.20 ml/L. Anyminimum value can be combined with any maximum value to define asuitable range. In a preferred embodiment, the concentration of a thecell protectant, such as polyethylene glycol in the culture medium isbetween 0.01 ml/L to 0.0.50, most preferably about 0.25 ml/L. Theconcentration of cell protectant in the culture medium described hereinmay be based on the total volume of the culture medium.

In one embodiment, the pH of the second culture medium can range, forexample, between a minimal value of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, or 7.0 to a maximal value of 8.0, 7.9, 7.8, 7.7, 7.6,7.5, 7.4, 7.3, 7.2, or 7.1. Any minimum value can be combined with anymaximum value to define a suitable range. In a preferred embodiment, thepH of the second culture medium is between 6.0 to 8.0, more preferably,between 6.5 to 7.5. Most preferably, the pH is 7.0.

In a preferred embodiment, the second culture medium includes soyhydrolysate, yeast extract HY YEST 412, glucose, thiamphenicol, at pH 7.More preferably, the culture medium includes 30 g/L soy hydrolysate, 20g/L yeast extract HY YEST 412, 60 g/L glucose, 15 mg/L thiamphenicol, atpH 7.

In one embodiment, the culturing is carried out under anaerobicconditions. In one embodiment, the culturing steps of the methods of theinvention (both seed and fermentation) are carried out under anaerobicconditions, although aerobic conditions for either of these phases maybe used as well.

Approaches to anaerobic culture of bacteria, such as C. difficile, areknown in the art and can employ, for example, nitrogen gas or a mixtureof nitrogen and hydrogen gases. The gas may be bubbled through themedium (e.g., sparging) during fermentation or passed through the areaabove the liquid in a culture chamber (e.g., the chamber headspace).

Culturing of the C. difficile cell can be carried out in an anaerobicchamber at approximately 30±1° C., 31±1° C., 32±1° C., 33±1° C., 34±1°C., 35±1° C., 36±1° C., 37±1° C., 38±1° C., or 39±1° C., preferablyabout 37±1° C. The culturing can be carried out for times ranging, forexample, between a minimal value of 1, 2, 3, 4, 7, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours to a maximal valueof 9 days, 8 days, 7 days, 6, days, 5 days, 4 days, 3 days, 48 hours, or36 hours, or 24 hours. Any minimum value can be combined with anymaximum value to define a suitable range. In a preferred embodiment, theculturing of the cell occurs for 11 to 48 hours, most preferably about24 hours. In another embodiment, the culturing of the cell occurs for 5to 25 hours, 10 to 20 hours, preferably about 15 hours. Growth can bemonitored by measuring the optical density (O.D.) of the medium.

In one embodiment, the method further includes isolating C. difficiletoxins from said medium. C. difficile toxins can be isolated and/orpurified from cultures using known protein purification methods. Thepurified toxins can then, for example, be inactivated by chemicallyinactivation treatment.

In an alternative aspect, the invention relates to a method of culturingC. difficile. The method includes culturing a C. difficile cell in amonoclonal antibody medium.

In another aspect, the invention relates to a method for production of aC. difficile toxin. The method includes culturing a C. difficilebacterium in a medium, as described above, under conditions to permitproduction of the toxin. The method further includes isolating the C.difficile toxin from the medium.

In one embodiment, the method includes culturing a C. difficile cell ina first medium under conditions that facilitate growth of C. difficile;inoculating a second medium with all or a portion of said first mediumafter said culturing; culturing said inoculated second medium underconditions that facilitate growth of C. difficile and toxin production;and isolating a C. difficile toxin from said second medium. In oneembodiment, culturing of the first or second media including C.difficile is carried out under anaerobic conditions.

In one embodiment, the C. difficile cell is cultured in a continuousculture system. In another embodiment, the invention relates to a methodof culturing C. difficile in a perfusion culture. Surprisingly, theinventors discovered that a recombinant C. difficile cell expressing amutant toxin may be successfully cultured in an anaerobic continuosculture and in an anaerobic perfusion culture. An advantage of acontinuous system and/or a perfusion system is that fresh media may beadded continuously. In addition, toxin byproducts from production may beremoved during production while maintaining cell viability in thesystem.

The continuous culture system may include providing fresh medium to thecells while simultaneously removing spent medium and cells from thebioreactor. The continuous culture may include a perfusion culture, inwhich the liquid outflow contains culture medium that is substantiallyfree of cells or includes a substantially lower cell concentration thanthat in the bioreactor. In a perfusion culture, cells can be retainedby, for example, filtration, ultrasonic filtration, centrifugation, orsedimentation.

In one embodiment, the spent media is removed and filtered, whichprevents cells from being removed from the bioreactor. The filter may bea cross-flow filter and/or a tangential flow filter. In one embodiment,said filtration system comprises a hollow fiber filter. In anotherembodiment, the cells are prevented from being removed from thebioreactor by a centrifugation step. In another embodiment, the cellsare prevented from being removed from the bioreactor by an ultrasonicfiltration step. In another embodiment, the cells are prevented frombeing removed from the bioreactor via a sedimentation system. In anotherembodiment, said filtration system comprises a flat-sheet cassette.

In yet another embodiment, the perfusion system comprises a hollow fiberfilter that will retain cells, but not the desired product. The cellsare recycled back into the bioreactor and the spent media containing thedesired product is passed through a desired molecular weight cut-offfilter. The filter will retain the desired product. Waste products notretained by the filter can be disposed or recycled.

Method of Producing a Mutant C. difficile Toxin

In one aspect, the invention relates to a method of producing a mutantC. difficile toxin. In one embodiment, the method includes culturing anyrecombinant cell or progeny thereof described above, under suitableconditions to express a polypeptide. The method further includes a stepof isolating the toxin from the medium.

In another embodiment, the method includes culturing a recombinant cellor progeny thereof under suitable conditions to express a polynucleotideencoding a mutant C. difficile toxin, wherein the cell includes thepolynucleotide encoding the mutant C. difficile toxin, and wherein themutant includes a glucosyltransferase domain having at least onemutation and a cysteine protease domain having at least one mutation,relative to the corresponding wild-type Clostridium difficile toxin. Inone embodiment, the cell lacks an endogenous polynucleotide encoding atoxin.

In a further embodiment, the method includes culturing a recombinant C.difficile cell or progeny thereof under suitable conditions to express apolynucleotide encoding a mutant C. difficile toxin, wherein the cellincludes the polynucleotide encoding the mutant C. difficile toxin andthe cell lacks an endogenous polynucleotide encoding a C. difficiletoxin.

In another aspect, the invention relates to a method of producing amutant C. difficile toxin. The method includes the steps of: (a)contacting a C. difficile cell with a recombinant Escherichia coli cell,wherein the C. difficile cell lacks an endogenous polynucleotideencoding a C. difficile toxin and the E. coli cell includes apolynucleotide that encodes a mutant C. difficile toxin; (b) culturingthe C. difficile cell and the E. coli cell under suitable conditions fortransfer of the polynucleotide from the E. coli cell to the C. difficilecell; (c) selecting the C. difficile cell comprising the polynucleotideencoding the mutant C. difficile toxin; (d) culturing the C. difficilecell of step (c) under suitable conditions to express thepolynucleotide; and (e) isolating the mutant C. difficile toxin.

In the inventive method, the recombinant E. coli cell includes aheterologous polynucleotide that encodes the mutant C. difficile toxin,described herein. The polynucleotide may be DNA or RNA. In one exemplaryembodiment, the polynucleotide that encodes the mutant C. difficiletoxin is codon-optimized for E. coli codon usage. Methods forcodon-optimizing a polynucleotide are known in the art.

In one embodiment, the polynucleotide includes a nucleic acid sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolynucleotide encoding a mutant C. difficile TcdA, as described above.In one embodiment, the polynucleotide includes a nucleic acid sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolynucleotide encoding a polypeptide having any one sequence selectedfrom SEQ ID NO: 1 to SEQ ID NO: 761. An exemplary polynucleotideencoding a mutant C. difficile toxin A includes SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 44, and SEQ ID NO: 45.

In another embodiment, the polynucleotide includes a nucleic acidsequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a polynucleotide encoding a mutant C. difficile TcdB, asdescribed above. An exemplary polynucleotide encoding a mutant C.difficile toxin B includes SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 46,and SEQ ID NO: 47. In another embodiment, the polynucleotide encodes SEQID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

In one embodiment, the E. coli cell that includes the heterologouspolynucleotide is an E. coli cell that stably hosts the heterologouspolynucleotide, which encodes the mutant C. difficile toxin. ExemplaryE. coli cells include a cell selected from the group consisting of MAXEfficiency® Stbl2™ E. coli Competent Cells (Invitrogen, Carlsbad,Calif.), One Shot® Stbl3™ Chemically Competent E. coli (Invitrogen,Carlsbad, Calif.), ElectroMAX™ Stbl4™ E. coli Competent Cells(Invitrogen, and E. coli CA434. In a preferred embodiment, the E. colicloning host cell is not DH5α. More preferably, the E. coli cloning hostcell is a MAX Efficiency® Stbl2™ E. coli Competent Cell.

The inventive method further includes a step of culturing the C.difficile cell and the E. coli cell under suitable conditions fortransfer of the polynucleotide from the E. coli cell to the C. difficilecell, resulting in a recombinant C. difficile cell. In a preferredembodiment, the culture conditions are suitable for transfer of thepolynucleotide from the E. coli cell (the donor cell) into the C.difficile cell (the recipient cell), and resulting in a geneticallystable inheritance.

Most preferably, the culture conditions are suitable for bacterialconjugation, which are known in the art. “Conjugation” refers to aparticular process of transferring a polynucleotide in which aunidirectional transfer of a polynucleotide (e.g., from a bacterialplasmid) occurs from one bacterium cell (i.e., the “donor”) to another(i.e., the “recipient”). The conjugation process involves donorcell-to-recipient cell contact. Preferably, the donor E. coli cell is anE. coli CA434 cell.

Exemplary suitable (conjugation) conditions for transferring of thepolynucleotide from the E. coli cell to the C. difficile cell includegrowing liquid cultures of C. difficile in brain heart infusion broth(BHI; Oxoid) or Schaedlers anaerobic broth (SAB; Oxoid). In anotherembodiment, solid C. difficile cultures may be grown on fresh blood agar(FBA) or BHI agar. Preferably, the C. difficile is grown at 37° C. in ananaerobic environment (e.g., 80% N₂, 10% CO₂, and 10% H₂ [vol/vol]). Inone embodiment, the suitable condition includes growing the E. coliaerobically in Luria-Bertani (LB) broth or on LB agar at 37° C. Forconjugative transfer to C. difficile, an exemplary suitable conditionincludes growing E. coli anaerobically on FBA. Antibiotics may beincluded in the liquid and solid media as is known in the art. Examplesof such antibiotics include cycloserine (250 μg/ml), cefoxitin (8μg/ml), chloramphenicol (12.5 μg/ml), thiamphenicol (15 μg/ml), anderythromycin (5 μg/ml).

The inventive method additionally includes a step of selecting theresulting recombinant C. difficile cell that includes the polynucleotideencoding the mutant C. difficile toxin. In an exemplary embodiment, therecombinant C. difficile cell is a recipient of the polynucleotideencoding the mutant C. difficile toxin from the recombinant E. coli cellvia conjugation.

The inventive method includes a step of culturing the recombinant cellor progeny thereof under suitable conditions to express thepolynucleotide encoding the mutant C. difficile toxin, resulting inproduction of a mutant C. difficile toxin. Suitable conditions for arecombinant cell to express the polynucleotide include cultureconditions suitable for growing a C. difficile cell, which are known inthe art. For example, suitable conditions may include culturing the C.difficile transformants in brain heart infusion broth (BHI; Oxoid) orSchaedlers anaerobic broth (SAB; Oxoid). In another embodiment, solid C.difficile cultures may be grown on FBA or BHI agar. Preferably, the C.difficile is grown at 37° C. in an anaerobic environment (e.g., 80% N₂,10% CO₂, and 10% H₂ [vol/vol]).

In one embodiment, the inventive method includes a step of isolating theresulting mutant C. difficile toxin. Methods of isolating a protein fromC. difficile are known in the art.

In another embodiment, the method includes a step of purifying theresulting mutant C. difficile toxin. Methods of purifying a polypeptide,such as chromatography, are known in the art.

In an exemplary embodiment, the method further includes a step ofcontacting the isolated mutant Clostridium difficile toxin with achemical crosslinking agent described above. Preferably, the agentincludes formaldehyde, ethyl-3-(3-dimethylaminopropyl) carbodiimide, ora combination of EDC and NHS. Exemplary reaction conditions aredescribed above and in the Examples section below.

In another aspect, the invention relates to an immunogenic compositionincluding a mutant C. difficile toxin described herein, produced by anymethod, preferably by any of the methods described above.

Antibodies

Surprisingly, the inventive immunogenic compositions described aboveelicited novel antibodies in vivo, suggesting that the immunogeniccompositions include a preserved native structure (e.g., a preservedantigenic epitope) of the respective wild-type C. difficile toxin andthat the immunogenic compositions include an epitope. The antibodiesproduced against a toxin from one strain of C. difficile may be capableof binding to a corresponding toxin produced by another strain of C.difficile. That is, the antibodies and binding fragments thereof may by“cross-reactive,” which refers to the ability to react with similarantigenic sites on toxins produced from multiple C. difficile strains.Cross-reactivity also includes the ability of an antibody to react withor bind an antigen that did not stimulate its production, i.e., thereaction between an antigen and an antibody that was generated against adifferent but similar antigen.

In one aspect, the inventors surprisingly discovered monoclonalantibodies having a neutralizing effect on C. difficile toxins, andmethods of producing the same. The inventive antibodies can neutralizeC. difficile toxin cytotoxicity in vitro, inhibit binding of C.difficile toxin to mammalian cells, and/or can neutralize C. difficiletoxin enterotoxicity in vivo. The present invention also relates toisolated polynucleotides that include nucleic acid sequences encodingany of the foregoing. In addition, the present invention relates to useof any of the foregoing compositions to treat, prevent, decrease therisk of, decrease severity of, decrease occurrences of, and/or delay theoutset of a C. difficile infection, C. difficile associated disease,syndrome, condition, symptom, and/or complication thereof in a mammal,as compared to a mammal to which the composition is not administered, aswell as methods for preparing said compositions.

The inventors further discovered that a combination of at least two ofthe neutralizing monoclonal antibodies can exhibit an unexpectedlysynergistic effect in respective neutralization of TcdA or TcdB.Anti-toxin antibodies or binding fragments thereof can be useful in theinhibition of a C. difficile infection.

An “antibody” is a protein including at least one or two heavy (H) chainvariable regions (abbreviated herein as VH), and at least one or twolight (L) chain variable regions (abbreviated herein as VL). The VH andVL regions can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (“CDR”), interspersed withregions that are more conserved, termed “framework regions” (FR). Theextent of the framework region and CDRs has been precisely defined (see,Kabat, E. A., et al. Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242, 1991, and Chothia, C. et al., J. Mol. Biol.196:901-917, 1987). The term “antibody” includes intact immunoglobulinsof types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), whereinthe light chains of the immunoglobulin may be of types kappa or lambda.

The antibody molecules can be full-length (e.g., an IgG1 or IgG4antibody). The antibodies can be of the various isotypes, including: IgG(e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In onepreferred embodiment, the antibody is an IgG isotype, e.g., IgG1. Inanother preferred embodiment, the antibody is an IgE antibody.

In another embodiment, the antibody molecule includes an“antigen-binding fragment” or “binding fragment,” as used herein, whichrefers to a portion of an antibody that specifically binds to a toxin ofC. difficile (e.g., toxin A). The binding fragment is, for example, amolecule in which one or more immunoglobulin chains is not full length,but which specifically binds to a toxin.

Examples of binding portions encompassed within the term “bindingfragment” of an antibody include (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CHI domains; (iv) a Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature 341:544-546, 1989), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR) having sufficientframework to specifically bind, e.g., an antigen binding portion of avariable region.

A binding fragment of a light chain variable region and a bindingfragment of a heavy chain variable region, e.g., the two domains of theFv fragment, VL and VH, can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv); see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also encompassed withinthe term “binding fragment” of an antibody. These antibody portions areobtained using techniques known in the art, and the portions arescreened for utility in the same manner as are intact antibodies.

As used herein, an antibody that “specifically binds” to or is“specific” for a particular polypeptide or an epitope on a particularpolypeptide is an antibody that binds to that particular polypeptide orepitope on a particular polypeptide without substantially binding to anyother polypeptide or polypeptide epitope. For example, when referring toa biomolecule (e.g., protein, nucleic acid, antibody, etc.) that“specifically binds” to a target, the biomolecule binds to its targetmolecule and does not bind in a significant amount to other molecules ina heterogeneous population of molecules that include the target, asmeasured under designated conditions (e.g. immunoassay conditions in thecase of an antibody). The binding reaction between the antibody and itstarget is determinative of the presence of the target in theheterogeneous population of molecules. For example, “specific binding”or “specifically binds” refers to the ability of an antibody or bindingfragment thereof to bind to a wild-type and/or mutant toxin of C.difficile with an affinity that is at least two-fold greater than itsaffinity for a non-specific antigen.

In an exemplary embodiment, the antibody is a chimeric antibody. Achimeric antibody can be produced by recombinant DNA techniques known inthe art. For example, a gene encoding the Fc constant region of a murine(or other species) monoclonal antibody molecule can be digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted. A chimeric antibody can also be created by recombinant DNAtechniques where DNA encoding murine variable regions can be ligated toDNA encoding the human constant regions.

In another exemplary embodiment, the antibody or binding fragmentthereof is humanized by methods known in the art. For example, oncemurine antibodies are obtained, a CDR of the antibody may be replacedwith at least a portion of a human CDR. Humanized antibodies can also begenerated by replacing sequences of the murine Fv variable region thatare not directly involved in antigen binding with equivalent sequencesfrom human Fv variable regions. General methods for generating humanizedantibodies are known in the art.

For example, monoclonal antibodies directed toward C. difficile TcdA orC. difficile TcdB can also be produced by standard techniques, such as ahybridoma technique (see, e.g., Kohler and Milstein, 1975, Nature, 256:495-497). Briefly, an immortal cell line is fused to a lymphocyte from amammal immunized with C. difficile TcdA, C. difficile TcdB, or a mutantC. difficile toxin described herein, and the culture supernatants of theresulting hybridoma cells are screened to identify a hybridoma producinga monoclonal antibody that binds to C. difficile TcdA or C. difficileTcdB. Typically, the immortal cell line is derived from the samemammalian species as the lymphocytes. Hybridoma cells producing amonoclonal antibody of the invention are detected by screening thehybridoma culture supernatants for antibodies that bind C. difficileTcdA or C. difficile TcdB using an assay, such as ELISA. Humanhybridomas can be prepared in a similar way.

As an alternative to producing antibodies by immunization and selection,antibodies of the invention may also be identified by screening arecombinant combinatorial immunoglobulin library with a C. difficileTcdA, C. difficile TcdB, or a mutant C. difficile toxin describedherein. The recombinant antibody library may be an scFv library or anFab library, for example. Moreover, the inventive antibodies describedherein may be used in competitive binding studies to identify additionalanti-TcdA or anti-TcdB antibodies and binding fragments thereof. Forexample, additional anti-TcdA or anti-TcdB antibodies and bindingfragments thereof may be identified by screening a human antibodylibrary and identifying molecules within the library that competes withthe inventive antibodies described herein in a competitive bindingassay.

In addition, antibodies encompassed by the present invention includerecombinant antibodies that may be generated by using phage displaymethods known in the art. In phage display methods, phage can be used todisplay antigen binding domains expressed from a repertoire or antibodylibrary (e.g., human or murine). Phage expressing an antigen bindingdomain that binds to an immunogen described herein (e.g., a mutant C.difficile toxin) can be selected or identified with antigen, e.g., usinglabeled antigen.

Also within the scope of the invention are antibodies and bindingfragments thereof in which specific amino acids have been substituted,deleted, or added. In particular, preferred antibodies have amino acidsubstitutions in the framework region, such as to improve binding to theantigen. For example, a selected, small number of acceptor frameworkresidues of the immunoglobulin chain can be replaced by thecorresponding donor amino acids. Preferred locations of thesubstitutions include amino acid residues adjacent to the CDR, or whichare capable of interacting with a CDR. Criteria for selecting aminoacids from the donor are described in U.S. Pat. No. 5,585,089 (e.g.,columns 12-16). The acceptor framework can be a mature human antibodyframework sequence or a consensus sequence.

As used herein, a “neutralizing antibody or binding fragment thereof”refers to a respective antibody or binding fragment thereof that bindsto a pathogen (e.g., a C. difficile TcdA or TcdB) and reduces theinfectivity and/or an activity of the pathogen (e.g., reducescytotoxicity) in a mammal and/or in cell culture, as compared to thepathogen under identical conditions in the absence of the neutralizingantibody or binding fragment thereof. In one embodiment, theneutralizing antibody or binding fragment thereof is capable ofneutralizing at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or moreof a biological activity of the pathogen, as compared to the biologicalactivity of the pathogen under identical conditions in the absence ofthe neutralizing antibody or binding fragment thereof.

As used herein, the term “anti-toxin antibody or binding fragmentthereof” refers to an antibody or binding fragment thereof that binds tothe respective C. difficile toxin (e.g., a C. difficile toxin A or toxinB). For example, an anti-toxin A antibody or binding fragment thereofrefers to an antibody or binding fragment thereof that binds to TcdA.

The antibodies or binding fragments thereof described herein may beraised in any mammal, wild-type and/or transgenic, including, forexample, mice, humans, rabbits, and goats.

When an immunogenic composition described above is one that has beenpreviously administered to a population, such as for vaccination, theantibody response generated in the subjects can be used to neutralizetoxins from the same strain and from a strain that did not stimulateproduction of the antibody. See, for example, Example 37, which showsstudies relating to cross-reactivity, generated by the immunogeniccomposition, between the 630 strain and toxins from various wild-type C.difficile strains.

In one aspect, the invention relates to an antibody or binding fragmentthereof specific to C. difficile TcdA. Monoclonal antibodies thatspecifically bind to TcdA include A65-33; A60-22; A80-29 and/or,preferably, A3-25.

In one aspect, the invention relates to an antibody or binding fragmentthereof specific to a TcdA from any wild type C. difficile strain, suchas those described above, e.g., to SEQ ID NO: 1. In another aspect, theinvention relates to an antibody or binding fragment thereof specific toan immunogenic composition described above. For example, in oneembodiment, the antibody or binding fragment thereof is specific to animmunogenic composition that includes SEQ ID NO: 4 or SEQ ID NO: 7. Inanother embodiment, the antibody or binding fragment thereof is specificto an immunogenic composition that includes SEQ ID NO: 4 or SEQ ID NO:7, wherein at least one amino acid of SEQ ID NO: 4 or SEQ ID NO: 7 iscrosslinked by formaldehyde, EDC, NHS, or a combination of EDC and NHS.In another embodiment, the antibody or binding fragment thereof isspecific to an immunogenic composition that includes SEQ ID NO: 84 orSEQ ID NO: 83.

Antibodies or binding fragments thereof having a variable heavy chainand variable light chain regions that are at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%or most preferably about 100% identity to the variable heavy and lightchain regions of A65-33; A60-22; A80-29 and/or, preferably, A3-25 canalso bind to TcdA.

In one embodiment, the antibody or antigen binding fragment thereofincludes a variable heavy chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable heavy chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 37.

In another embodiment, the antibody or antigen binding fragment thereofincludes a variable light chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable light chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 36.

In yet a further aspect, the antibody or antigen binding fragmentthereof includes a variable heavy chain region including an amino acidsequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto a variable heavy chain region amino acid sequence set forth in SEQ IDNO: 37, and a variable light chain region including an amino acidsequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto a variable light chain region amino acid sequence set forth in SEQ IDNO: 36.

In another embodiment, antibodies or binding fragments thereof havingcomplementarity determining regions (CDRs) of variable heavy chainsand/or variable light chains of A65-33; A60-22; A80-29 and/or,preferably, A3-25 can also bind to TcdA. The CDRs of the variable heavychain region of A3-25 are shown in Table 4, below.

TABLE 4 Variable Heavy Chain CDR Amino Acid Sequences Clone Chain CDRAmino Acid Sequence SEQ ID NO: A3-25 Heavy CDR1 GFTFTNYWMN 41 CDR2EIRLKSHNYATHFAESVKG 42 CDR3 DYYGNPAFVY 43

The CDRs of the variable light chain region of A3-25 are shown in Table5, below.

TABLE 5 Variable Light Chain CDR Amino Acid Sequences Clone Chain CDRAmino Acid Sequence SEQ ID NO: A3-25 Light CDR1 RSSQSLIHSNGNTYLH 38 CDR2KVSNRFS 39 CDR3 SQTTYFPYT 40

In one embodiment, the antibody or binding fragment thereof includesamino acid sequences of the heavy chain complementarity determiningregions (CDRs) as shown in SEQ ID NOs: 41 (CDR H1), 42 (CDR H2) and 43(CDR H3), and/or the amino acid sequences of the light chain CDRs asshown in SEQ ID NOs: 38 (CDR L1), 39 (CDR L2) and 40 (CDR L3).

In one exemplary embodiment, the antibody or binding fragment thereofspecific to C. difficile toxin A specifically binds to an epitope withinthe N-terminal region of TcdA e.g., an epitope between amino acids1-1256 of a TcdA, according to the numbering of SEQ ID NO: 1.

In a preferred embodiment, the antibody or binding fragment thereofspecific to C. difficile toxin A specifically binds to an epitope withinthe C-terminal region of toxin A, e.g., an epitope between amino acids1832 to 2710 of a TcdA, according to the numbering of SEQ ID NO: 1.Examples include A3-25; A65-33; A60-22; A80-29.

In yet another embodiment, the antibody or binding fragment thereofspecific to C. difficile toxin A specifically binds to an epitope withinthe “translocation” region of C. difficile toxin A, e.g., an epitopethat preferably includes residues 956-1128 of a TcdA, according to thenumbering of SEQ ID NO: 1, such as an epitope between amino acids659-1832 of a TcdA, according to the numbering of SEQ ID NO: 1.

In another aspect, the invention relates to an antibody or bindingfragment thereof specific to C. difficile TcdB. For example, theantibody or binding fragment thereof may be specific to a TcdB from anywild type C. difficile strain, such as those described above, e.g., toSEQ ID NO: 2. In another aspect, the invention relates to an antibody orbinding fragment thereof specific to an immunogenic compositiondescribed above. For example, in one embodiment, the antibody or bindingfragment thereof is specific to an immunogenic composition that includesSEQ ID NO: 6 or SEQ ID NO: 8.

In another embodiment, the antibody or binding fragment thereof isspecific to an immunogenic composition that includes SEQ ID NO: 6 or SEQID NO: 8, wherein at least one amino acid of SEQ ID NO: 6 or SEQ ID NO:8 is crosslinked by formaldehyde, EDC, NHS, or a combination of EDC andNHS. In another embodiment, the antibody or binding fragment thereof isspecific to an immunogenic composition that includes SEQ ID NO: 86 orSEQ ID NO: 85.

Monoclonal antibodies that specifically bind to TcdB include antibodiesproduced by the B2-31; B5-40, B70-2; B6-30; B9-30; B59-3; B60-2; B56-6;and/or, preferably, B8-26 clones described herein.

Antibodies or binding fragments thereof that can also bind to TcdBinclude those having a variable heavy chain and variable light chainregions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,preferably about 98%, more preferably about 99% or most preferably about100% identity to the variable heavy and light chain regions of B2-31;B5-40, B70-2; B6-30; B9-30; B59-3; B60-2; B56-6, preferably B8-26,B59-3, and/or B9-30.

In one embodiment, the antibody or antigen binding fragment thereofincludes a variable heavy chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable heavy chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 49.

In one embodiment, the antibody or antigen binding fragment thereofincludes a variable heavy chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable heavy chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 60.

In one embodiment, the antibody or antigen binding fragment thereofincludes a variable heavy chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable heavy chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 71.

In another embodiment, the antibody or antigen binding fragment thereofincludes a variable light chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable light chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 55.

In another embodiment, the antibody or antigen binding fragment thereofincludes a variable light chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable light chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 66.

In another embodiment, the antibody or antigen binding fragment thereofincludes a variable light chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable light chain region amino acid sequence of A3-25 as set forth inSEQ ID NO: 77.

The amino acid sequence for the variable heavy chain of a neutralizingantibody of C. difficile TcdB (B8-26 mAb) is set forth in SEQ ID NO: 49.See Table 25-a.

TABLE 25-a Variable Heavy Chain Amino Acid Sequences SEQ ID  CloneRegion Amino Acid Sequence NO: B8-26 Signal MGWSCIILFLVATATGVHS 50peptide Variable QVQLQQPGAELVKPGA 49 heavy PVKLSCKAS GYSFTSYWMN chainWVKQRPGRGLEWIG RIDPSNSEIYYNQKF KDKATLTVDKSSSTAYIQLSSLTSEDSAVYYCAS GHYGSIFAY WGQGTTLTVSS CDR1 GYSFTSYWMN 51 CDR2RIDPSNSEIYYNQKF 52 CDR3 GHYGSIFAY 53 Constant AKTTPPSVYPLAPGNSK 54region (IgG1)

The amino acid sequence for the variable light chain of a neutralizingantibody of C. difficile TcdB (B8-26 mAb) is set forth in SEQ ID NO: 55.See Table 25-b.

TABLE25-b Variable Light (κ) Chain Amino Acid Sequences Clone RegionAmino Acid Sequence SEQ ID NO: B8-26 Signal MRFQVQVLGLLLLWISGAQCD 56peptide Variable VQITQSPSYLAASPGETITINC 55 light RASKSISKYLA WYQEKPGKTNKchain LLLYSGSTLQS GIPSRFSGS RSGTDFTLIISSLEPEDSAMYYC QQHNEYPLTFGAGTKLELKRADAAPTVSIFPPSSEEFQ CDR1 RASKSISKYLA 57 CDR2 SGSTLQS 58 CDR3QQHNEYPLT 59

In one embodiment, the antibody or binding fragment thereof includesamino acid sequences of the heavy chain CDRs as shown in SEQ ID NOs: 51(CDR H1), 52 (CDR H2) and 53 (CDR H3), and/or the amino acid sequencesof the light chain CDRs as shown in SEQ ID NOs: 57 (CDR L1), 58 (CDR L2)and 59 (CDR L3).

The amino acid sequence for the variable heavy chain of a neutralizingantibody of C. difficile TcdB (B59-3 mAb) is set forth in SEQ ID NO: 60.See Table 26-a.

TABLE 26-a Variable Heavy Chain Amino Acid Sequences Clone RegionAmino Acid Sequence SEQ ID NO: B59-3 Signal MGWSYIILFLVATATDVHS 61peptide Variable QVQLQQPGAELVKPGASVKLS 60 heavy CKAS GYTFTSYWMH chainWVKQRPGQGLEWIG VINPSNGRSTYSEKF KTTATVTVDKSSSTAYMQL SILTSEDSAVYYCARAYYSTSYYAMDY WGQGTSVTVSS CDR1 GYTFTSYWMH 62 CDR2 VINPSNGRSTYSEKF 63 CDR3AYYSTSYYAMDY 64 Constant AKTTPPSVYPLAPGNSK 65 region (IgG1)

The amino acid sequence for the variable light chain of a neutralizingantibody of C. difficile TcdB (B59-3 mAb) is set forth in SEQ ID NO: 66.See Table 26-b.

TABLE 26-b Variable Light (κ) Chain Amino Acid Sequences Clone RegionAmino Acid Sequence SEQ ID NO: B59-3 Signal MKLPVRLLVLMFWIPASSSD 67peptide Variable VLMTQSPLSLPVSLGDQASIS 66 light C RSSQNIVHSNGNTYLE chainWYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTYFTLKISRVE AEDLGVYYCFQGSHFPFTFGTGTKLEIKRADAAPTVSIFPPSSEEFQ CDR1 RSSQNIVHSNGNTYLE 68 CDR2 KVSNRFS 69 CDR3FQGSHFPFT 70

In one embodiment, the antibody or binding fragment thereof includesamino acid sequences of the heavy chain CDRs as shown in SEQ ID NOs: 62(CDR H1), 63 (CDR H2) and 64 (CDR H3), and/or the amino acid sequencesof the light chain CDRs as shown in SEQ ID NOs: 68 (CDR L1), 69 (CDR L2)and 70 (CDR L3).

The amino acid sequence for the variable heavy chain of a neutralizingantibody of C. difficile TcdB (B9-30 mAb) is set forth in SEQ ID NO: 71.See Table 27-a.

TABLE 27-a Variable Heavy Chain Amino Acid Sequences Clone RegionAmino Acid Sequence SEQ ID NO: B9-30 Signal MGWSCIILFLVATATGVHS 72peptide Variable QVQLQQPGAEVVKPGAPVKLS 71 heavy CKAS GYPFTNYWMN chainWVKQRPGRGLEWIG RIDPSNSEIYYNQKFKDKATLTV DKSSSTAYIQLSSLTSEDSAVYYCAS GHYGSIFAY WGQGTTLTVSS CDR1 GYPFTNYWMN 73 CDR2 RIDPSNSEIYYNQKF 74CDR3 GHYGSIFAY 75 Constant AKTTPPSVYPLAPGNSK 76 region (IgG1)

The amino acid sequence for the variable light chain of a neutralizingantibody of C. difficile TcdB (B9-30 mAb) is set forth in SEQ ID NO: 77.See Table 27-b.

TABLE 27-b Variable Light (κ) Chain Amino Acid Sequences Clone RegionAmino Acid Sequence SEQ ID NO: B9-30 Signal MRFQVQVLGLLLLWISGAQCD 78peptide Variable VQITQSPSYLAASPGETITINC 77 light RASKSISKYLA WYQEKPGKTNchain KLLIYSGSTLQS GIPSRFSGS RSGTDFTLIISSLEPEDSAMYYC QQHNEYPLTFGAGTKLELKRADAAPTVSIFPPSSEEFQ CDR1 RASKSISKYLA 79 CDR2 SGSTLQS 80 CDR3QQHNEYPLT 81

In one embodiment, the antibody or binding fragment thereof includesamino acid sequences of the heavy chain CDRs as shown in SEQ ID NOs: 73(CDR H1), 74 (CDR H2) and 75 (CDR H3), and/or the amino acid sequencesof the light chain CDRs as shown in SEQ ID NOs: 79 (CDR L1), 80 (CDR L2)and 81 (CDR L3).

In one aspect, the invention relates to an antibody or binding fragmentthereof specific to a wild type C. difficile TcdB from any C. difficilestrain, such as those described above, e.g., to SEQ ID NO: 2. In anotheraspect, the invention relates to an antibody or binding fragment thereofspecific to an immunogenic composition described above. For example, inone embodiment, the antibody or binding fragment thereof is specific toan immunogenic composition that includes SEQ ID NO: 6 or SEQ ID NO: 8.In another embodiment, the antibody or binding fragment thereof isspecific to an immunogenic composition that includes SEQ ID NO: 6 or SEQID NO: 8, wherein at least one amino acid of SEQ ID NO: 6 or SEQ ID NO:8 is crosslinked by formaldehyde, EDC, NHS, or a combination of EDC andNHS.

Antibodies or binding fragments thereof having a variable heavy chainand variable light chain regions that are at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%or most preferably about 100% identity to the variable heavy and lightchain regions of B2-31; B5-40, B70-2; B6-30; B9-30; B59-3; B60-2; B56-6;and/or, preferably, B8-26 can also bind to TcdB.

In one embodiment, the antibody or antigen binding fragment thereofincludes a variable heavy chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable heavy chain region amino acid sequence of B8-26 (SEQ ID NO:49).

In another embodiment, the antibody or antigen binding fragment thereofincludes a variable light chain region including an amino acid sequenceat least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to avariable light chain region amino acid sequence of B8-26 (SEQ ID NO:55).

In yet a further aspect, the antibody or antigen binding fragmentthereof includes a variable heavy chain region including an amino acidsequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto a variable heavy chain region amino acid sequence of B8-26 (SEQ IDNO: 49), and a variable light chain region including an amino acidsequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto a variable light chain region amino acid sequence of B8-26 (SEQ IDNO: 55).

In another embodiment, antibodies or binding fragments thereof havingCDRs of variable heavy chains and/or variable light chains of B2-31;B5-40, B70-2; B6-30; B9-30; B59-3; B60-2; B56-6; and/or, preferably,B8-26 can also bind to TcdB.

In one embodiment, the antibody or binding fragment thereof includesamino acid sequences of the heavy chain complementarity determiningregions (CDRs) of B8-26, and/or the amino acid sequences of the lightchain CDRs of B8-26.

In a preferred embodiment, the antibody or binding fragment thereofspecific to C. difficile toxin B specifically binds to an epitope withinthe N-terminal region of toxin B, e.g., an epitope between amino acids1-1256 of a TcdB, according to the numbering of SEQ ID NO: 2. Examplesinclude B2-31; B5-40; B8-26; B70-2; B6-30; and B9-30.

In an exemplary embodiment, the antibody or binding fragment thereofspecific to C. difficile toxin B specifically binds to an epitope withinthe C-terminal region of toxin B, e.g., an epitope between amino acids1832 to 2710 of a TcdB, according to the numbering of SEQ ID NO: 2.

In yet another embodiment, the antibody or binding fragment thereofspecific to C. difficile toxin B specifically binds to an epitope withinthe “translocation” region of C. difficile toxin B, e.g., an epitopethat preferably includes residues 956-1128 of a TcdB, according to thenumbering of SEQ ID NO: 2, such as an epitope between amino acids659-1832 of a TcdB. Examples include B59-3; B60-2; and B56-6.

Combinations of Antibodies

The anti-toxin antibody or binding fragment thereof can be administeredin combination with other anti-C. difficile toxin antibodies (e.g.,other monoclonal antibodies, polyclonal gamma-globulin) or a bindingfragment thereof. Combinations that can be used include an anti-toxin Aantibody or binding fragment thereof and an anti-toxin B antibody orbinding fragment thereof.

In another embodiment, a combination includes an anti-toxin A antibodyor binding fragment thereof and another anti-toxin A antibody or bindingfragment thereof. Preferably, the combination includes a neutralizinganti-toxin A monoclonal antibody or binding fragment thereof and anotherneutralizing anti-toxin A monoclonal antibody or binding fragmentthereof. Surprisingly, the inventors discovered that such a combinationresulted in a synergistic effect in neutralization of toxin Acytotoxicity. For example, the combination includes a combination of atleast two of the following neutralizing anti-toxin A monoclonalantibodies: A3-25; A65-33; A60-22; and A80-29. More preferably, thecombination includes A3-25 antibody and at least one of the followingneutralizing anti-toxin A monoclonal antibodies: A65-33; A60-22; andA80-29. Most preferably, the combination includes all four antibodies:A3-25; A65-33; A60-22; and A80-29.

In a further embodiment, a combination includes an anti-toxin B antibodyor binding fragment thereof and another anti-toxin B antibody or bindingfragment thereof. Preferably, the combination includes a neutralizinganti-toxin B monoclonal antibody or binding fragment thereof and anotherneutralizing anti-toxin B monoclonal antibody or binding fragmentthereof. Surprisingly, the inventors discovered that such a combinationresulted in a synergistic effect in neutralization of toxin Bcytotoxicity. More preferably, the combination includes a combination ofat least two of the following neutralizing anti-toxin B monoclonalantibodies: B8-26; B9-30 and B59-3. Most preferably, the combinationincludes all three antibodies: B8-26; B9-30 and B59-3.

In yet another embodiment, a combination includes an anti-toxin Bantibody or binding fragment thereof and another anti-toxin B antibodyor binding fragment thereof. As stated previously, the inventorsdiscovered that a combination of at least two of the neutralizingmonoclonal antibodies can exhibit an unexpectedly synergistic effect inrespective neutralization of toxin A and toxin B.

In another embodiment, the agents of the invention can be formulated asa mixture, or chemically or genetically linked using art recognizedtechniques thereby resulting in covalently linked antibodies (orcovalently linked antibody fragments), having both anti-toxin A andanti-toxin B binding properties. The combined formulation may be guidedby a determination of one or more parameters such as the affinity,avidity, or biological efficacy of the agent alone or in combinationwith another agent.

Such combination therapies are preferably additive and/or synergistic intheir therapeutic activity, e.g., in the inhibition, prevention (e.g.,of relapse), and/or treatment of C. difficile-related diseases ordisorders. Administering such combination therapies can decrease thedosage of the therapeutic agent (e.g., antibody or antibody fragmentmixture, or cross-linked or genetically fused bispecific antibody orantibody fragment) needed to achieve the desired effect.

It is understood that any of the inventive compositions, for example, ananti-toxin A and/or anti-toxin B antibody or binding fragment thereof,can be combined in different ratios or amounts for therapeutic effect.For example, the anti-toxin A and anti-toxin B antibody or respectivebinding fragment thereof can be present in a composition at a ratio inthe range of 0.1:10 to 10:0.1, A:B. In another embodiment, theanti-toxin A and anti-toxin B antibody or respective binding fragmentthereof can be present in a composition at a ratio in the range of0.1:10 to 10:0.1, B:A.

In another aspect, the invention relates to a method of producing aneutralizing antibody against a C. difficile TcdA. The method includesadministering an immunogenic composition as described above to a mammal,and recovering the antibody from the mammal. In a preferred embodiment,the immunogenic composition includes a mutant C. difficile TcdA havingSEQ ID NO: 4, wherein at least one amino acid of the mutant C. difficileTcdA is chemically crosslinked, preferably by formaldehyde or1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Exemplary neutralizingantibodies against TcdA that may be produced include A65-33; A60-22;A80-29 and/or A3-25.

In yet another aspect, the invention relates to a method of producing aneutralizing antibody against a C. difficile TcdB. The method includesadministering an immunogenic composition as described above to a mammal,and recovering the antibody from the mammal. In a preferred embodiment,the immunogenic composition includes a mutant C. difficile TcdB havingSEQ ID NO: 6, wherein at least one amino acid of the mutant C. difficileTcdB is chemically crosslinked, preferably by formaldehyde or1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Exemplary neutralizingantibodies against TcdB that may be produced include B2-31; B5-40,B70-2; B6-30; B9-30; B59-3; B60-2; B56-6; and/or B8-26.

Formulations

Compositions of the present invention (such as, e.g., compositionsincluding a mutant C. difficile toxin, immunogenic compositions,antibodies and/or antibody binding fragments thereof described herein)may be in a variety of forms. These include, for example, semi-solid andsolid dosage forms, suppositories, liquid forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, liposomes, and/or dried form, such as, for example,lyophilized powder form, freeze-dried form, spray-dried form, and/orfoam-dried form. For suppositories, binders and carriers include, forexample, polyalkylene glycols or triglycerides; such suppositories canbe formed from mixtures containing the inventive compositions. In anexemplary embodiment, the composition is in a form that is suitable forsolution in, or suspension in, liquid vehicles prior to injection. Inanother exemplary embodiment, the composition is emulsified orencapsulated in liposomes or microparticles, such as polylactide,polyglycolide, or copolymer.

In a preferred embodiment, the composition is lyophilized andextemporaneously reconstituted prior to use.

In one aspect, the present invention relates to pharmaceuticalcompositions that include any of the compositions described herein (suchas, e.g., compositions including a mutant C. difficile toxin,immunogenic compositions, antibodies and/or antibody binding fragmentsthereof described herein), formulated together with a pharmaceuticallyacceptable carrier. “Pharmaceutically acceptable carriers” include anysolvents, dispersion media, stabilizers, diluents, and/or buffers thatare physiologically suitable.

Exemplary stabilizers include carbohydrates, such as sorbitol, mannitol,starch, dextran, sucrose, trehalose, lactose, and/or glucose; inertproteins, such as albumin and/or casein; and/or other large, slowlymetabolized macromolecules, such as polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized SEPHAROSE™ agarose, agarose, cellulose, etc/), aminoacids, polymeric amino acids, amino acid copolymers, and lipidaggregates (such as oil droplets or liposomes). Additionally, thesecarriers may function as immunostimulating agents (i.e., adjuvants).

Preferably, the composition includes trehalose. Preferred amounts oftrehalose (% by weight) include from a minimum of about 1%, 2%, 3%, or4% to a maximum of about 10%, 9%, 8%, 7%, 6%, or 5%. Any minimum valuecan be combined with any maximum value to define a suitable range. Inone embodiment, the composition includes about 3%-6% trehalose, mostpreferably, 4.5% trehalose, for example, per 0.5 mL dose.

Examples of suitable diluents include distilled water, saline,physiological phosphate-buffered saline, glycerol, alcohol (such asethanol), Ringer's solutions, dextrose solution, Hanks' balanced saltsolutions, and/or a lyophilization excipient.

Exemplary buffers include phosphate (such as potassium phosphate, sodiumphosphate); acetate (such as sodium acetate); succinate (such as sodiumsuccinate); glycine; histidine; carbonate,Tris(tris(hydroxymethyl)aminomethane), and/or bicarbonate (such asammonium bicarbonate) buffers. Preferably, the composition includes trisbuffer. Preferred amounts of tris buffer include from a minimum of about1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM to a maximum of about 100 mM,50 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, or11 mM. Any minimum value can be combined with any maximum value todefine a suitable range. In one embodiment, the composition includesabout 8 mM to 12 mM tris buffer, most preferably, 10 mM tris buffer, forexample, per 0.5 mL dose.

In another preferred embodiment, the composition includes histidinebuffer. Preferred amounts of histidine buffer include from a minimum ofabout 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM to a maximum of about100 mM, 50 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM,12 mM, or 11 mM. Any minimum value can be combined with any maximumvalue to define a suitable range. In one embodiment, the compositionincludes about 8 mM to 12 mM histidine buffer, most preferably, 10 mMhistidine buffer, for example, per 0.5 mL dose.

In yet another preferred embodiment, the composition includes phosphatebuffer. Preferred amounts of phosphate buffer include from a minimum ofabout 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM to a maximum of about100 mM, 50 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM,12 mM, or 11 mM. Any minimum value can be combined with any maximumvalue to define a suitable range. In one embodiment, the compositionincludes about 8 mM to 12 mM phosphate buffer, most preferably, 10 mMphosphate buffer, for example, per 0.5 mL dose.

The pH of the buffer will generally be chosen to stabilize the activematerial of choice, and can be ascertainable by those in the art byknown methods. Preferably, the pH of the buffer will be in the range ofphysiological pH. Thus, preferred pH ranges are from about 3 to about 8;more preferably, from about 6.0 to about 8.0; yet more preferably, fromabout 6.5 to about 7.5; and most preferably, at about 7.0 to about 7.2.

In some embodiments, the pharmaceutical compositions may include asurfactant. Any surfactant is suitable, whether it is amphoteric,non-ionic, cationic or anionic. Exemplary surfactants include thepolyoxyethylene sorbitan esters surfactants (e.g., TWEEN®), such aspolysorbate 20 and/or polysorbate 80; polyoxyethylene fatty ethersderived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brijsurfactants), such as triethyleneglycol monolauryl ether (Brij 30);Triton X 100, or t-octylphenoxypolyethoxyethanol; and sorbitan esters(commonly known as the SPANs), such as sorbitan trioleate (Span 85) andsorbitan monolaurate, and combinations thereof. Preferred surfactantsinclude polysorbate 80 (polyoxyethylene sorbitan monooleate).

Preferred amounts of polysorbate 80 (% by weight) include from a minimumof about 0.001%, 0.005%, or 0.01%, to a maximum of about 0.010%, 0.015%,0.025%, or 1.0%. Any minimum value can be combined with any maximumvalue to define a suitable range. In one embodiment, the compositionincludes about 0.005%-0.015% polysorbate 80, most preferably, 0.01%polysorbate 80.

In an exemplary embodiment, the immunogenic composition includestrehalose and phosphate 80. In another exemplary embodiment, theimmunogenic composition includes tris buffer and polysorbate 80. Inanother exemplary embodiment, the immunogenic composition includeshistidine buffer and polysorbate 80. In yet another exemplaryembodiment, the immunogenic composition includes phosphate buffer andpolysorbate 80.

In one exemplary embodiment, the immunogenic composition includestrehalose, tris buffer and polysorbate 80. In another exemplaryembodiment, the immunogenic composition includes trehalose, histidinebuffer and polysorbate 80. In yet another exemplary embodiment, theimmunogenic composition includes trehalose, phosphate buffer andpolysorbate 80.

The compositions described herein may further include components ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, and/or mineral oil. Examples include glycols such aspropylene glycol or polyethylene glycol.

In some embodiments, the pharmaceutical composition further includesformaldehyde. For example, in a preferred embodiment, a pharmaceuticalcomposition that further includes formaldehyde has an immunogeniccomposition, wherein the mutant C. difficile toxin of the immunogeniccomposition has been contacted with a chemical crosslinking agent thatincludes formaldehyde. The amount of formaldehyde present in thepharmaceutical composition may vary from a minimum of about 0.001%,0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.010%,0.013%, or 0.015%, to a maximum of about 0.020%, 0.019%, 0.018%, 0.017%0.016%, 0.015%, 0.014%, 0.013%, 0.012% 0.011% or 0.010%. Any minimumvalue can be combined with any maximum value to define a suitable range.In one embodiment, the pharmaceutical composition includes about 0.010%formaldehyde.

In some alternative embodiments, the pharmaceutical compositionsdescribed herein do not include formaldehyde. For example, in apreferred embodiment, a pharmaceutical composition that does not includeformaldehyde has an immunogenic composition, wherein at least one aminoacid of the mutant C. difficile toxin is chemically crosslinked by anagent that includes EDC. More preferably, in such an embodiment, themutant C. difficile toxin has not been contacted with a chemicalcrosslinking agent that includes formaldehyde. As another exemplaryembodiment, a pharmaceutical composition that is in a lyophilized formdoes not include formaldehyde.

In another embodiment, the compositions described herein may include anadjuvant, as described below. Preferred adjuvants augment the intrinsicimmune response to an immunogen without causing conformational changesin the immunogen that may affect the qualitative form of the immuneresponse.

Exemplary adjuvants include 3 De-O-acylated monophosphoryl lipid A(MPL™) (see GB 2220211 (GSK)); an aluminum hydroxide gel such asAlhydrogel™ (Brenntag Biosector, Denmark); aluminum salts (such asaluminum hydroxide, aluminum phosphate, aluminum sulfate), which may beused with or without an immunostimulating agent such as MPL or 3-DMP,QS-21, polymeric or monomeric amino acids such as polyglutamic acid orpolylysine.

Yet another exemplary adjuvant is an immunostimulatory oligonucleotidesuch as a CpG oligonucleotide (see, e.g., WO 1998/040100,WO2010/067262), or a saponin and an immunostimulatory oligonucleotide,such as a CpG oligonucleotide (see, e.g., WO 00/062800). In a preferredembodiment, the adjuvant is a CpG oligonucleotide, most preferably a CpGoligodeoxynucleotides (CpG ODN). Preferred CpG ODN are of the B Classthat preferentially activate B cells. In aspects of the invention, theCpG ODN has the nucleic acid sequence 5′T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3′ (SEQ ID NO: 48) wherein *indicates a phosphorothioate linkage. The CpG ODN of this sequence isknown as CpG 24555, which is described in WO2010/067262. In a preferredembodiment, CpG 24555 is used together with an aluminium hydroxide saltsuch as Alhydrogel.

A further class of exemplary adjuvants include saponin adjuvants, suchas Stimulon™ (QS-21, which is a triterpene glycoside or saponin, Aquila,Framingham, Mass.) or particles generated therefrom such as ISCOMs(immune stimulating complexes) and ISCOMATRIX® adjuvant. Accordingly,the compositions of the present invention may be delivered in the formof ISCOMs, ISCOMS containing CTB, liposomes or encapsulated in compoundssuch as acrylates or poly(DL-lactide-co-glycoside) to form microspheresof a size suited to adsorption. Typically, the term “ISCOM” refers toimmunogenic complexes formed between glycosides, such as triterpenoidsaponins (particularly Quil A), and antigens which contain a hydrophobicregion. In a preferred embodiment, the adjuvant is an ISCOMATRIXadjuvant.

Other exemplary adjuvants include RC-529, GM-CSF and Complete Freund'sAdjuvant (CFA) and Incomplete Freund's Adjuvant (IFA).

Yet another class of exemplary adjuvants is glycolipid analoguesincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates,each of which is substituted in the sugar residue by an amino acid.

Optionally, the pharmaceutical composition includes two or moredifferent adjuvants. Preferred combinations of adjuvants include anycombination of adjuvants including, for example, at least two of thefollowing adjuvants: alum, MPL, QS-21, ISCOMATRIX, CpG, and Alhydrogel.An exemplary combination of adjuvants includes a combination of CpG andAlhydrogel.

Alternatively, in one embodiment, the composition is administered to themammal in the absence of an adjuvant.

Compositions described herein can be administered by any route ofadministration, such as, for example, parenteral, topical, intravenous,mucosal, oral, subcutaneous, intraarterial, intracranial, intrathecal,intraperitoneal, intranasal, intramuscular, intradermal, infusion,rectal, and/or transdermal routes for prophylactic and/or therapeuticapplications. In a preferred embodiment, the route of administration ofthe composition is parenteral, more preferably, intramuscularadministration. Typical intramuscular administration is performed in thearm or leg muscles.

Compositions described herein can be administered in combination withtherapies that are at least partly effective in prevention and/ortreatment of C. difficile infection. For example, a composition of theinvention may be administered before, concurrently with, or afterbiotherapy; probiotic therapy; stool implants; immunotherapy (such asintravenous immunoglobulin); and/or an accepted standard of care for theantibiotic treatment of C. difficile associated disease (CDAD), such asmetronidazole and/or vancomycin.

A composition of the present invention relating to toxin A and toxin Bmay be administered to the mammal in any combination. For example, animmunogenic composition including a mutant C. difficile TcdA may beadministered to the mammal before, concurrently with, or afteradministration of an immunogenic composition including a mutant C.difficile TcdB. Conversely, an immunogenic composition including amutant C. difficile TcdB may be administered to the mammal before,concurrently with, or after administration of an immunogenic compositionincluding a mutant C. difficile TcdA.

In another embodiment, a composition including an anti-toxin A antibodyor binding fragment thereof may be administered to the mammal before,concurrently with, or after administration of a composition including ananti-toxin B antibody or binding fragment thereof. Conversely, acomposition including an anti-toxin B antibody or binding fragmentthereof may be administered to the mammal before, concurrently with, orafter administration of a composition including an anti-toxin A antibodyor binding fragment thereof.

In a further embodiment, a composition of the present invention may beadministered to the mammal before, concurrently with, or afteradministration of a pharmaceutically acceptable carrier. For example, anadjuvant may be administered before, concurrently with, or afteradministration of a composition including a mutant C. difficile toxin.Accordingly, a composition of the present invention and apharmaceutically acceptable carrier can be packaged in the same vial orthey can be packaged in separate vials and mixed before use. Thecompositions can be formulated for single dose administration and/ormultiple dose administration.

Methods of Protecting and/or Treating C. difficile Infection in a Mammal

In one aspect, the invention relates to a method of inducing an immuneresponse to a C. difficile toxin in a mammal. The method includesadministering an effective amount of a composition described herein tothe mammal. For example, the method may include administering an amounteffective to generate an immune response to the respective C. difficiletoxin in the mammal.

In an exemplary embodiment, the invention relates to a method ofinducing an immune response to a C. difficile TcdA in a mammal. Themethod includes administering an effective amount of an immunogeniccomposition that includes a mutant C. difficile TcdA to the mammal. Inanother exemplary embodiment, the invention relates to a method ofinducing an immune response to a C. difficile TcdB in a mammal. Themethod includes administering an effective amount of an immunogeniccomposition that includes a mutant C. difficile TcdB to the mammal.

In a further embodiment, the method includes administering an effectiveamount of an immunogenic composition that includes a mutant C. difficileTcdA and an effective amount of an immunogenic composition that includesa mutant C. difficile TcdB to the mammal. In additional aspects, thecompositions described herein may be used to treat, prevent, decreaserisk of, decrease severity of, decrease occurrences of, and/or delayoutset of a C. difficile infection, C. difficile associated disease,syndrome, condition, symptom, and/or complication thereof in a mammal,as compared to a mammal to which the composition is not administered.The method includes administering an effective amount of the compositionto the mammal.

Three clinical syndromes caused by C. difficile infection arerecognized, based on the severity of the infection. The most severe formis pseudomembranous colitis (PMC), which is characterized by profusediarrhea, abdominal pain, systemic signs of illness, and a distinctiveendoscopic appearance of the colon.

Antibiotic-associated colitis (AAC) is also characterized by profusediarrhea, abdominal pain and tenderness, systemic signs (e.g., fever),and leukocytosis. Intestinal injury in AAC is less severe than in PMC,the characteristic endoscopic appearance of the colon in PMC is absent,and mortality is low.

Finally, antibiotic-associated diarrhea (AAD, which is also known as C.difficile associated diarrhea (CDAD) is a relatively mild syndrome, andis characterized by mild to moderate diarrhea, lacking both largeintestinal inflammation (as characterized by, e.g., abdominal pain andtenderness) and systemic signs of infection (e.g., fever).

These three distinct syndromes typically occur in an increasing order offrequency. That is, PMC typically occurs less frequently than AAC, andAAD is typically the most frequent clinical presentation of C. difficiledisease.

A frequent complication of C. difficile infection is recurrent orrelapsing disease, which occurs in up to 20% of all subjects who recoverfrom C. difficile disease. Relapse may be characterized clinically asAAD, AAC, or PMC. Patients who relapse once are more likely to relapseagain.

As used herein, conditions of a C. difficile infection include, forexample, mild, mild-to-moderate, moderate, and severe C. difficileinfection. A condition of C. difficile infection may vary depending onpresence and/or severity of symptoms of the infection.

Symptoms of a C. difficile infection may include physiological,biochemical, histologic and/or behavioral symptoms such as, for example,diarrhea; colitis; colitis with cramps, fever, fecal leukocytes, andinflammation on colonic biopsy; pseudomembranous colitis;hypoalbuminemia; anasarca; leukocytosis; sepsis; abdominal pain;asymptomatic carriage; and/or complications and intermediatepathological phenotypes present during development of the infection, andcombinations thereof, etc. Accordingly, for example, administration ofan effective amount of the compositions described herein may, forexample treat, prevent, decrease risk of, decrease severity of, decreaseoccurrences of, and/or delay outset of diarrhea; abdominal pain, cramps,fever, inflammation on colonic biopsy, hypoalbuminemia, anasarca,leukocytosis, sepsis, and/or asymptomatic carriage, etc., as compared toa mammal to which the composition was not administered.

Risk factors of a C. difficile infection may include, for example,present or immediate future use of an antimicrobial (any antimicrobialagent with an antibacterial spectrum and/or activity against anaerobicbacteria are encompassed, including, for example, antibiotics that causedisruption of normal colonic microbiota, e.g., clindamycin,cephalosporins, metronidazole, vancomycin, fluoroquinolones (includinglevofloxacin, moxifloxacin, gatifloxacin, and ciprofloxacin), linezolid,etc.); present or immediate future withdrawal of prescribedmetronidazole or vancomycin; present or immediate future admission to ahealthcare facility (such as a hospital, chronic care facility, etc.)and healthcare workers; present or immediate future treatment withproton-pump inhibitors, H2 antagonists, and/or methotrexate, or acombination thereof; present or risk of gastrointestinal diseases, suchas inflammatory bowel disease; past, present, or immediate futuregastrointestinal surgery or gastrointestinal procedure on the mammal;past or present recurrence of a C. difficile infection and/or a CDAD,e.g., patients who have had a C. difficile infection and/or a CDAD onceor more than once; and humans aged at least about 65 and above.

In the methods described herein, the mammal may be any mammal, such as,for example, mice, hamsters, primates, and humans. In a preferredembodiment, the mammal is a human. According to the present invention,the human may include individuals who have exhibited a C. difficileinfection, C. difficile associated disease, syndrome, condition,symptom, and/or complication thereof; individuals who are presentlyexhibiting a C. difficile infection, C. difficile associated disease,syndrome, condition, symptom, and/or complication thereof; andindividuals who are at risk of a C. difficile infection, C. difficileassociated disease, syndrome, condition, symptom, and/or complicationthereof.

Examples of individuals who have shown symptoms of C. difficileinfection include individuals who have shown or are showing symptomsdescribed above; individuals who have had or are having a C. difficileinfection and/or a C. difficile associated disease (CDAD); andindividuals who have a recurrence of a C. difficile infection and/orCDAD.

Examples of patients who are at risk of a C. difficile infection includeindividuals at risk of or are presently undergoing planned antimicrobialuse; individuals at risk of or are presently undergoing withdrawal ofprescribed metronidazole or vancomycin; individuals who are at risk ofor are presently undergoing a planned admission to a healthcare facility(such as a hospital, chronic care facility, etc.) and healthcareworkers; and/or individuals at risk of or are presently undergoing aplanned treatment with proton-pump inhibitors, H2 antagonists, and/ormethotrexate, or a combination thereof; individuals who have had or areundergoing gastrointestinal diseases, such as inflammatory boweldisease; individuals who have had or are undergoing gastrointestinalsurgery or gastrointestinal procedures; and individuals who have had orare having a recurrence of a C. difficile infection and/or a CDAD, e.g.,patients who have had a C. difficile infection and/or a CDAD once ormore than once; individuals who are about 65 years old or older. Suchat-risk patients may or may not be presently showing symptoms of a C.difficile infection.

In asymptomatic patients, prophylaxis and/or treatment can begin at anyage (e.g., at about 10, 20, or 30 years old). In one embodiment,however, it is not necessary to begin treatment until a patient reachesat least about 45, 55, 65, 75, or 85 years old. For example, thecompositions described herein may be administered to an asymptomatichuman who is aged 50-85 years.

In one embodiment, the method of preventing, decreasing risk of,decreasing severity of, decreasing occurrences of, and/or delayingoutset of a C. difficile infection, C. difficile associated disease,syndrome, condition, symptom, and/or complication thereof in a mammalincludes administering an effective amount of a composition describedherein to a mammal in need thereof, a mammal at risk of, and/or a mammalsusceptible to a C. difficile infection. An effective amount includes,for example, an amount sufficient to prevent, decrease risk of, decreaseseverity of, decrease occurrences of, and/or delay outset of a C.difficile infection, C. difficile associated disease, syndrome,condition, symptom, and/or complication thereof in a mammal, as comparedto a mammal to which the composition is not administered. Administrationof an effective amount of the compositions described herein may, forexample, prevent, decrease risk of, decrease severity of, decreaseoccurrences of, and/or delay outset of diarrhea; abdominal pain, cramps,fever, inflammation on colonic biopsy, hypoalbuminemia, anasarca,leukocytosis, sepsis, and/or asymptomatic carriage, etc., as compared toa mammal to which the composition was not administered. In a preferredembodiment, the method includes administering an effective amount of animmunogenic composition described herein to the mammal in need thereof,the mammal at risk of, and/or the mammal susceptible to a C. difficileinfection.

In an additional embodiment, the method of treating, decreasing severityof, and/or delaying outset of a C. difficile infection, C. difficileassociated disease, syndrome, condition, symptom, and/or complicationthereof in a mammal includes administering an effective amount of acomposition described herein to a mammal suspected of, or presentlysuffering from a C. difficile infection. An effective amount includes,for example, an amount sufficient to treat, decrease severity of, and/ordelay the outset of a C. difficile infection, C. difficile associateddisease, syndrome, condition, symptom, and/or complication thereof in amammal, as compared to a mammal to which the composition is notadministered.

Administration of an effective amount of the composition may improve atleast one sign or symptom of C. difficile infection in the subject, suchas those described below. Administration of an effective amount of thecompositions described herein may, for example, decrease severity ofand/or decrease occurrences of diarrhea; decrease severity of and/ordecrease occurrences of abdominal pain, cramps, fever, inflammation oncolonic biopsy, hypoalbuminemia, anasarca, leukocytosis, sepsis, and/orasymptomatic carriage, etc., as compared to a mammal to which thecomposition was not administered. Optionally, the presence of symptoms,signs, and/or risk factors of an infection is determined beforebeginning treatment. In a preferred embodiment, the method includesadministering an effective amount of an antibody and/or binding fragmentthereof described herein to the mammal suspected of, or presentlysuffering from, a C. difficile infection.

Accordingly, an effective amount of the composition refers to an amountsufficient to achieve a desired effect (e.g., prophylactic and/ortherapeutic effect) in the methods of the present invention. Forexample, the amount of an immunogen for administration may vary from aminimum of about 1 μg, 5 μg, 25 μg, 50 μg, 75 μg, 100 μg, 200 μg, 500μg, or 1 mg to a maximum of about 2 mg, 1 mg, 500 μg, 200 μg perinjection. Any minimum value can be combined with any maximum value todefine a suitable range. Typically about 10, 20, 50 or 100 μg perimmunogen is used for each human injection.

The amount of a composition of the invention administered to the subjectmay depend on the type and severity of the infection and/or on thecharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. It may also depend on the degree,severity, and type of disease. An effective amount may also varydepending upon factors, such as route of administration, target site,physiological state of the patient, age of the patient, whether thepatient is human or an animal, other therapies administered, and whethertreatment is prophylactic or therapeutic. The skilled artisan will beable to determine appropriate amounts depending on these and otherfactors.

An effective amount may include one effective dose or multiple effectivedoses (such as, for example, 2, 3, 4 doses, or more) for use in themethods herein. Effective dosages may need to be titrated to optimizesafety and efficacy.

A combination of amount and frequency of dose adequate to accomplishprophylactic and/or therapeutic uses is defined as a prophylatically- ortherapeutically-effective regimen. In a prophylactic and/or therapeuticregimen, the composition is typically administered in more than onedosage until a sufficient immune response has been achieved. Typically,the immune response is monitored and repeated dosages are given if theimmune response starts to wane.

The compositions may be administered in multiple dosages over a periodof time. Treatment can be monitored by assaying antibody, or activatedT-cell or B-cell responses to the therapeutic agent (e.g., theimmunogenic composition including a mutant C. difficile toxin) overtime. If the response falls, a booster dosage is indicated.

EXAMPLES Example 1 Identification of Toxin-Negative C. difficile Strains{TC “Identification of Toxin-Negative C. difficile Strains” \f C \I “3”}

To identify C. difficile strains lacking toxin (A and B) genes and toxinexpression, 13 C. difficile strains were tested. Culture media of 13 C.difficile strains were tested by ELISA for toxin A. Seven strainsexpressed toxin A: C. difficile 14797-2, C. difficile 630, C. difficileBDMS, C. difficile W1194, C. difficile 870, C. difficile 1253, and C.difficile 2149. See FIG. 3.

Six strains did not express toxin A and lacked the entire pathogenicitylocus: C. difficile 1351 (ATCC 43593™), C. difficile 3232 (ATCCBAA-1801™), C. difficile 7322 (ATCC 43601™), C. difficile 5036 (ATCC43603™), C. difficile 4811 (4 ATCC 3602™), and C. difficile VPI 11186(ATCC 700057™). VPI 11186 was selected based upon its effectiveness totake up plasmid DNA by conjugation.

The same 13 strains were tested in a multiplex PCR assay using primersoutside of the pathogenicity locus (PaLoc; Braun et al., Gene. 1996 Nov.28; 181(1-2):29-38.). The PCR results demonstrated the DNA from the 6toxin A negative strains by ELISA did not amplify any genes from thePaLoc (tcdA-tcdE). The PaLoc flanking sequences (cdd3 and cdu2) werepresent (data not shown).

Example 2 Inactivation of Sporulation Pathway in C. difficile VPI 11186

Knocking-out the spore-forming function of the C. difficile productionstrain facilitates large scale fermentation in a safe manufacturingenvironment. The ClosTron system was used to create an asporogenic C.difficile strain. See Heap et al., J Microbiol Methods. 2009 July;78(1):79-85. The ClosTron system allows targeted gene inactivation witha group II intron for site directed insertional inactivation of a spo0A1clostridial gene. The toxin-minus production strain VPI11186 wassubjected to sporulation inactivation by the ClosTron technology.Erythromycin resistant mutants were selected and the presence of theinsertional cassette was confirmed by PCR (not shown). The inability oftwo independent clones to form spores was confirmed.

Example 3 Genetic Modification of Toxin A and B Genes to InactivateCytotoxicity Function

Full-length mutant toxins A and B open reading frames (ORFs) based onstrain 6304 genome sequences were designed for custom synthesis at BlueHeron Biotech. See, for example, SEQ ID NOs: 9-14. The active site forthe glucosyltransferase activity responsible for cellular toxicity wasaltered by two allelic substitutions: D285A/D287A (see SEQ ID NO: 3) fortoxin A, and D286A/D288A (see SEQ ID NO: 5) for toxin B. Two nucleotideswere mutated in each aspartate (D) codon to create the codon for alanine(A). See, for example, SEQ ID NOs: 9-14. In addition, a pair of vectorsexpressing mutant toxins lacking cysteine residues was constructedfollowing custom synthesis at Blue Heron Biotech. Seven cysteineresidues from mutant toxin A and 9 cysteine residues from mutant toxin Bwere replaced with alanine. The substitutions include catalyticcysteines of the A and B toxin autocatalytic protease. Also, silentmutations were introduced where necessary to eliminate restrictionenzyme sites used for vector construction.

Example 4 pMTL84121fdx Expression Vector

The plasmid shuttle vector used for C. difficile mutant toxin antigenexpression was selected from the pMTL8000-series modular systemdeveloped by the Minton lab (see Heap et al., J Microbiol Methods. 2009July; 78(1):79-85). The chosen vector pMTL84121fdx contains the C.difficile plasmid pCD6 Gram+replicon, the catP(chloramphenicol/thiamphenicol) selectable marker, the p15aGram-replicon and tra function, and the C. sporogenes feredoxin promoter(fdx) and distal multiple cloning site (MCS). Empirical data suggestedthat the low-copy number p15a replicon conferred greater stability in E.coli than the ColE1 alternative. The fdx promoter was selected as ityielded higher expression than other promoters tested in experimentswith CAT reporter constructs (e.g. tcdA, tcdB; or heterologous tetR orxylR) (data not shown).

Example 5 Cloning the Modified Toxin ORFs into pMTL84121fdx

Full-length mutant toxin A and B open reading frames (ORFs) based onstrain 6304 genome sequences were subcloned using pMTL84121fdx vectormultiple cloning NdeI and BglII sites using standard molecular biologytechniques. To facilitate cloning, the ORFs were flanked by a proximalNdeI site containing the start codon and a BglII site just downstream ofthe stop codon.

Example 6 Site Directed Mutagenesis of TcdA to Create a Triple Mutant

The catalytic cysteine residue of the autocatalytic protease domain wassubstituted (i.e., C700A for TcdA and C698A for TcdB) in SEQ ID NOs: 3and 5, i.e., in each of the “double mutants.” For mutagenesis of mutanttoxin A, a 2.48 kb NdeI-HindIII fragment from the TcdA D285A/D287Aexpression plasmid was subcloned into pUC19 (cut with same) andsite-directed mutagenesis was performed on this template. Once the newalleles were confirmed by DNA sequence analysis, the modifiedNdeI-HindIII fragments were reintroduced into the expression vectorpMTL84121 fdx to create the “triple mutants,” i.e., SEQ ID NO: 4 and SEQID NO: 6.

Example 7 Site Directed Mutagenesis of TcdB to Create a Triple Mutant

For mutagenesis of mutant toxin B, a 3.29 kb NdeI-EcoNI fragment fromthe mutant toxin B plasmid was modified and reintroduced. As the EcoNIsite is not present in available cloning vectors a slightly larger 3.5kb NdeI-EcoRV fragment was subcloned into pUC19 (prepared withNdeI-SmaI). After mutagenesis, the modified internal 3.3 kb NdeI-EcoNIfragment was excised and used to replace the corresponding mutant toxinB expression vector pMTL84121 fdx fragment. As the cloning efficiency ofthis directional strategy was found to be quite low, an alternativestrategy for introducing the C698A allele involving replacement of a 1.5kb DraIII was attempted in parallel. Both strategies independentlyyielded the desired recombinants.

Example 8 Creating Additional Mutant Toxin Variants by Site-DirectedMutagenesis

At least twelve different C. difficile mutant toxin variants wereconstructed. Allelic substitutions were introduced into N-terminalmutant toxin gene fragments by site directed mutagenesis (Quickchange®kit). Recombinant toxins were also engineered as reference controls toevaluate the capacity of this plasmid-based system to generate proteinquantitatively equivalent in biological activity to native toxinspurified from wild-type C. difficile strains. In this case, allelicsubstitutions were introduced to revert the original glucosyltransferasesubstitutions. In addition, a pair of cysteineless mutant toxin vectorswas constructed following custom synthesis at Blue Heron Biotech.

The twelve toxin variants include (1) a mutant C. difficile toxin Ahaving a D285A/D287A mutation (SEQ ID NO: 3); (2) a mutant C. difficiletoxin B having a D286A/D288A mutation (SEQ ID NO: 5); (3) a mutant C.difficile toxin A having a D285A/D287A C700A mutation (SEQ ID NO: 4);(4) a mutant C. difficile toxin B having a D286A/D288A C698A mutation(SEQ ID NO: 6); (5) a recombinant toxin A having SEQ ID NO: 1; (6) arecombinant toxin B having SEQ ID NO: 2; (7) a mutant C. difficile toxinA having a C700A mutation; (8) a mutant C. difficile toxin B having aC698A mutation; (9) a mutant C. difficile toxin A having a C700A C597S,C1169S, C14075, C1623S, C2023S, and C2236S mutation; (10) a mutant C.difficile toxin B having a C698A C395S, C595S, C824S, C870S, C1167S,C1625S, C1687S, and C2232S mutation; (11) a mutant C. difficile toxin Ahaving a D285A, D287A, C700A, D269A, R272A, E460A, and R462A mutation(SEQ ID NO: 7); and (12) a mutant C. difficile toxin B having a D270A,R273A, D286A, D288A, D461A, K463A, and C698A mutation (SEQ ID NO: 8)

Penta mutant toxins were also constructed by site directed mutagenesisand by using the same materials and methods as described above, e.g., inExamples 1-7. The penta mutant for toxin B included the followingsubstitutions D286A/D288A C698A/E970K/E976K (SEQ ID NO: 184). The pentamutant toxin B was expressed in VPI 11186 spo0A-negative cells asdescribed above. A western blot using mAb# B8-26 (SEQ ID NO: 49), whichis specific to an N-terminal epitope of toxin B, was done to confirmexpression of the penta mutant toxin B. In the second, third, and fourthlanes from the left, 50 ng, 30 ng, and 10 ng of purified triple mutant B(SEQ ID NO: 86), respectively, were used as a reference protein. In thefifth and sixth lanes from the left, a 1:100 dilution and a 1:1000dilution, respectively, of the penta mutant toxin B cell lysateconcentrate was assessed. The estimated amount of protein in the pentamutant toxin B concentration is about 1000 ug/mL. As shown on the blot,the penta mutant toxin B (concentrated) exhibits a protein band ofexpected size at about 270 kD.

Example 9 Stability of Transformants

Rearranged plasmids were obtained with the commonly-used DH5 E. coli labstrain. In contrast, transformations using the Invitrogen Stbl2™ E. colihost yielded slow-growing full-length mutant toxin recombinants afterthree days of growth at 30° C. on LB chloramphenicol (25 μg/ml) plates.Lower cloning efficiencies were obtained with related Stbl3™ and Stbl4™E. coli strains, although these lines were found to be stable forplasmid maintenance. Transformants were subsequently propagated in agaror in liquid culture under chloramphenicol selection at 30° C. The useof LB (Miller's) media was also found to improve the recovery and growthof transformants compared with animal-free tryptone-soy based media.

Example 10 Transformation of C. difficile with pMTL84121 Fdx EncodingWild-Type or Genetic Mutant Toxin Genes

Transformation of C. difficile by E. coli conjugal transfer was doneessentially as described in Heap et al., Journal of MicrobiologicalMethods, 2009. 78(1): p. 79-85. E. coli host CA434 was transformed withpMTL84121 fdx encoding wild type or variant mutant toxin genes. E. colihost CA434 is the intermediate to mobilize expression plasmids into theC. difficile production strain VPI 11186 spo0A1. CA434 is a derivativeof E. coli HB101. This strain harbors the Tra+Mob+R702 conjugativeplasmid which confers resistance to Km, Tc, Su, Sm/Spe, and Hg (due toTn1831). Chemically competent or electrocompetent CA434 cells wereprepared and expression vector transformants were selected on Miller'sLB CAM plates at 30° C. Slow growing colonies appearing after 3 dayswere picked and amplified in 3 mL LB chloramphenicol cultures untilmid-log phase (˜24 h, 225 rpm, orbital shaker at 30° C.). E. colicultures were harvested by low speed (5,000 g) centrifugation to avoidbreaking pili, and cell pellets were resuspended gently with a wide-boretransfer pipette in 1 mL PBS. Cells were concentrated by low speedcentrifugation. Most of the PBS was removed by inversion and the drainedpellets were transferred into the anaerobic chamber and resuspended with0.2 mL of C. difficile culture, spotted onto BHIS agar plates and leftto grow for 8 h or overnight. In the case of mutant toxin Atransformants, better results were achieved with overnight conjugation.Cell patches were scraped into 0.5 mL PBS and 0.1 mL was plated on BHISselection media supplemented with 15 μg/mL thiamphenicol (more potentanalog of chloramphenicol) and D-cycloserine/cefoxitin to kill E. colidonor cells. Transformants appearing 16-24 h later were purified byre-streaking onto a new BHIS (plus supplements) plate and subsequentcultures were tested for expression of recombinant toxins or mutanttoxins. Glycerol permanents and seed stocks were prepared from clonesshowing good expression. Plasmid minipreps were also prepared from 2 mLcultures using a modified Qiagen kit procedure in which cells werepretreated with lysozyme (not essential). The C. difficile miniprep DNAwas used as a template for PCR sequencing to verify clone integrity.Alternatively, plasmid maxiprep DNA was prepared from E. coli Stbl2™transformants and sequenced.

Example 11 C. difficile Expression Analysis of the Toxin A and B TripleMutant (SEQ ID NOs: 4 and 6, Respectively) and Hepta B Mutant (SEQ IDNO: 8)

Transformants were grown either in 2 mL cultures (for routine analysis)or in vent-capped flasks (for time course experiments). Samples (2 mL)were centrifuged briefly (10,000 rpm, 30 s) to concentrate the cells:supernatants were decanted and concentrated 10× (Amicon-ultra 30k);pellets were drained and frozen at −80° C. Cell pellets were thawed onice, resuspended in 1 mL lysis buffer (Tris-HCl pH7.5; 1 mM EDTA, 15%glycerol) and sonicated (1×20 s burst with microtip). The lysate wascentrifuged at 4° C. and supernatant was concentrated 5-fold. Samples ofsupernatant and lysate were combined with sample buffer and heat treated(10 min, 80° C.) before loading onto duplicate 3-8% Tris-acetateSDS-PAGE gels (Invitrogen). One gel was stained with Coomassie, thesecond was electroblotted for western analysis. Toxin A-specific andToxin B-specific rabbit antisera (Fitgerald; Biodesign) were used todetect mutant toxin A and B variants. Expression of the hepta mutanttoxin B (SEQ ID NO: 8) was also confirmed by western blot hybridization.

Example 12 Abrogation of Glucosyltransferase Activity of the MutantToxins

Genetic double mutant (DM) toxins A and B (SEQ ID NOs: 3 and 5,respectively) and triple mutant (TM) toxins A and B (SEQ ID NOs: 4 and6, respectively) did not transfer ¹⁴C-glucose to 10 μg of RhoA, Rac1 andCdc42 GTPases in in vitro glucosylation assays in the presence ofUDP-¹⁴C-glucose [30 μM], 50 mM HEPES, pH 7.2, 100 mM KCl, 4 mM MgCl₂, 2mM MnCl₂, 1 mM DTT, and 0.1 μg/μL BSA. However, wild-type A and B toxincontrols (having SEQ ID NOs: 1 and 2, respectively) transferred¹⁴C-glucose to GTPases efficiently at a low dose of 10 and 1 ng each(and lower-data not shown) (FIGS. 4A and 4B), even in the presence of100 μg of mutant toxin (FIG. 4B) indicating at least 100,000-foldreduction compared to respective wild-type toxins. Similar results weredetected for Cdc42 GTPase (data not shown).

Specifically, in FIG. 4B, wild-type toxin A and toxin B (1 ng) or triplemutant toxin A and triple mutant toxin B (100 μg) were incubated withRhoA GTPase in the presence of UDP-¹⁴C-glucose for 2 hr at 30° C. Asillustrated, 1 ng of wild-type TcdA and TcdB transferred ¹⁴C-glucose toRhoA but 100 μg of triple mutant toxin A and triple mutant toxin B didnot. When 1 ng of wild-type TcdA or TcdB was spiked into the reactionwith respective 100 μg of triple mutant toxin A or triple mutant toxinB, glucosylation of RhoA was detected, indicating the lack ofglucosylation inhibitors. The sensitivity of detection for theglucosylation activity was established to be 1 ng of wild-type toxin ina background of 100 μg mutant toxin (ratio of 1:100,000). The resultsshow that the mutations in the active site of the glucosyltransferase inthe triple mutant toxin A and triple mutant toxin B reduced anymeasurable (less than 100,000-fold lower activity compared to theactivity of the respective wild-type toxins) glucosyltransferaseactivity. A similar assay was also developed to quantifyglucosyltransferase activity by TCA precipitation of glucosylatedGTPases.

Example 13 Abrogation of Auto-Catalytic Cysteine Protease Activity

The function of auto-catalytic cleavage was abrogated in the triplegenetic mutants A and B (TM) (SEQ ID NOs: 4 and 6, respectively) whenthe cysteine protease cleavage site was mutated. As illustrated in FIG.5, the wild type (wt) toxins A and B (SEQ ID NOs: 3 and 5, respectively)are cleaved in the presence of inositol-6-phosphate. The double mutanttoxins A and B (SEQ ID NOs: 3 and 5, respectively) are also cleaved inthe presence of inositol-6-phosphate (data not shown), similar to thatfor wild-type. Toxin A (SEQ ID NO: 3) is cleaved from 308 kDa into 2fragments of 245 and 60 kDa. Toxin B (SEQ ID NO: 5) is cleaved from 270kDa into two fragments of 207 and 63 kDa. The triple genetic mutants Aand B (TM) (SEQ ID NOs: 4 and 6, respectively) remain unaffected at 308and 270 kDa respectively, even in the presence of inositol-6-phosphate.See FIG. 5. Therefore, the cysteine protease activity was inactivated bygenetic modification.

More specifically, in FIG. 5, one μg of triple mutant A and triplemutant B were incubated for 90 minutes at room temperature (21±5° C.) inparallel with wild-type TcdA and TcdB from List Biologicals. Thecleavage reaction was performed in 20 μL volume in Tris-HCl, pH 7.5, 2mM DTT in the presence or absence of inositol-6-phosphate (10 mM forTcdA and 0.1 mM for Tcd B). The entire reaction volume was then loadedon a 3-8% SDS/PAGE; the protein bands were visualized by silverstaining. As illustrated, wt Tcd A and TcdB were cleaved into twoprotein bands of 245 kD and 60 kD and 207 kD and 63 kD, respectively, inthe presence of inositol-6-phosphate. The triple mutant toxin A andtriple mutant toxin B were not cleaved, thus confirming the C700Amutation in triple mutant toxin A and C698A mutation in triple mutanttoxin B blocked cleavage.

Example 14 Residual Cytotoxicity of Triple Mutant Toxins A and B (SEQ IDNOs: 4 and 6, Respectively)

The genetic mutant toxins were evaluated for their cytotoxicity by an invitro cytotoxicity assay in IMR90 cells, a human diploid lung fibroblastcell line. These cells are sensitive to both toxin A and B. As analternative preferred embodiment, Vero normal kidney cells fromCercopithecus aethiops may be used in the cytotoxicity assay since theywere observed to have reasonable sensitivities to toxin A and B.Preferably, HT-29 human colorectal adenocarcinoma cells are not used inthe cytotoxicity assay because they have shown significantly decreasedsensitivities to the toxins, as compared to the Vero and IMR90 celllines. See, for example, Table 6 below.

TABLE 6 Cell Line Sensitivities to Toxins A and B* Toxin EC₅₀ (pg/ml)Cell line 50 μg/ml Cells/well 48 hours 72 hours Vero A 10000 1816 244(ATCC CCL-81 ™) B 10000 62 29 IMR90 A 10000 1329 1152 (ATCC CCL-186TM) B10000 14 13 HT-29 A 10000 >1E6 >1E6 (ATCC HTB-38 ™) B 10000 11089 53313*In vitro cytotoxicity assay was performed by measuring cellular ATPusing luciferase-based substrate, CellTiter-Glo ® (Promega, Madison, WI)

Serially diluted genetic mutant toxin or wt toxin samples were added tothe cell monolayers grown in 96-well tissue culture plates. Afterincubation at 37° C. for 72 h, the plates were evaluated formetabolically active cells by measuring cellular ATP levels by additionof luciferase based CellTiterGlo® reagent (Promega, Madison, Wis.)generating luminescence expressed as relative luminescence units (RLUs).High RLUs show that the cells are viable, low RLUs show that the cellsare not metabolically active and are dying. The level of cytotoxicity,expressed as EC₅₀, is defined as the amount of wt toxin or geneticmutant toxin that elicits a 50% reduction in RLUs compared to levels incell culture controls (details of this assay are provided below). Asshown in FIG. 6, Tables 7A, and Table 8A, the EC₅₀ values of TcdA andTcdB were about 0.92 ng/mL and 0.009 ng/mL, respectively. The EC₅₀values of triple mutant toxin A and triple mutant toxin B were about8600 ng/mL and 74 ng/mL, respectively. Despite an approximate10,000-fold reduction in cytotoxicity relative to wt toxins, bothgenetic mutant toxins still demonstrated low residual levels ofcytotoxicity. This residual cytotoxicity could be blocked byneutralizing antitoxin monoclonal antibodies indicating that it wasspecific to the triple mutant toxins but not likely related to the knownenzymatic activities of the wt toxins (glucosylation orautoproteolysis).

Both wt toxins exhibit potent in vitro cytotoxicity, with small amountsof the toxins being sufficient to cause various effects on mammaliancells such as cell rounding (cytopathic effect or CPE) and lack ofmetabolic activity (as measured by ATP levels). Consequently, two invitro assays (a CPE or cell rounding assay and an ATP assay) have beendeveloped to verify that no residual cytotoxicity in the mutant toxindrug substances remains. The results are expressed as EC₅₀, which is theamount of toxin or mutant toxin that causes 1) 50% of the cells todevelop CPE or 2) 50% reduction in ATP levels as measured in relativelight units.

In the CPE assay, a sample of drug substance is serially diluted andincubated with IMR90 cells, which are observed for a potentialcytopathic effect. The CPE assay is scored on a scale of 0 (normalcells) to 4 (˜100% cell rounding) and a score of 2 (˜50% cell rounding)is defined as EC₅₀ value of the test sample. This method is used fortesting of mutant toxin drug substance at the concentration of 1000μg/mL, which is the maximal tolerable concentration that can be testedin this assay without matrix interference. Consequently, no detectablecytotoxicity is reported as EC₅₀>1000 μg/ml.

The ATP assay is based on measurement of the amount of luminescencesignal generated from ATP, which is proportional to the number ofmetabolically active cells. The maximal tolerable concentration that canbe tested in this assay without assay interference is about 200 μg/mL.Therefore no detectable cytotoxity in this assay is reported as EC₅₀>200μg/mL.

Different concentrations of mutant toxin A and B were added to cells inparallel with toxin controls. The endpoints of the assay were cellviability determined by cellular ATP levels using the CellTiter-Glo®(Promega). The degree of luminescence is proportional to ATP levels orviable cell number.

The in vitro cytotoxicity (EC₅₀) of wild type (wt) toxin A was 920 μg/mLand 9 μg/mL for toxin B. The in vitro cytotoxicity (EC₅₀) of mutanttoxin A (SEQ ID NO: 4) was 8600 ng/mL and 74 ng/mL for mutant toxin B(SEQ ID NO: 6). Although these values represent reductions of 9348 and8222-fold, respectively, residual cytotoxicity was detected in bothmutant toxins.

In other words, the cytotoxicity of triple mutant toxins A and B (SEQ IDNOs: 4 and 6, respectively) was significantly reduced in the in vitrocytotoxicity assay in IMR-90 cells relative to the cytotoxicity of wttoxins A and B (SEQ ID NOs: 1 and 2, respectively). As illustrated inFIG. 6, although both triple mutant toxins exhibited significantreduction in cytotoxicity (10⁴ fold) relative to the wt toxin, residualcytotoxicity was observed at higher concentrations of both triple mutanttoxins.

Furthermore, the residual cytotoxicity of each triple mutant toxin couldbe completely neutralized (e.g., at least a 6-8 log₁₀ reduction intoxicity, relative to the wild-type toxin toxicity) by the toxinspecific antibodies. See Example 16, below.

Cell culture assays are more sensitive for detection of cytotoxicitythan in vivo animal models. When delivered by either i.p. or i.v routesin the mouse lethal challenge model, the wt TcdA has an LD₅₀ of ˜50 ngper mouse while the wt TcdB is more potent with an LD₅₀ of ˜5 ng permouse. In contrast, the cell culture based in vitro assays describedabove have EC₅₀ values of 100 pg per well for wt TcdA and 2 pg per wellfor wt TcdB.

Example 15 Residual Cytotoxicity of the Genetic Hepta Mutant Toxin B(SEQ ID NO: 8), and Cytotoxicity of Penta Mutant Toxin B (SEQ ID NO:184)

As illustrated in FIG. 7, the EC₅₀ values are similar for the triplemutant toxin B (SEQ ID NO: 6) (20.78 ng/mL) and hepta mutant toxin B(SEQ ID NO: 8) (35.9 ng/mL) mutants indicating that the four additionalmutations to further modify the glucosyltransferase active site andGTPase substrate binding site did not further reduce the cytotoxicity ofthe genetic mutant toxins. The EC₅₀ values were also similar for thedouble mutant toxin B (SEQ ID NO: 5) as they are for the triple andhepta mutant toxins (data not shown). This observation suggests themechanism for cytotoxicity of the mutant toxins is surprisinglyindependent of the glucosyltransferase and substrate recognitionmechanism.

The penta mutant toxin B (SEQ ID NO: 184) was evaluated for itscytotoxicity by an in vitro ATP cytotoxicity assay in IMR90 cells, asdescribed above. See, e.g., Example 14. As shown in FIG. 26, thecytotoxicity of the penta mutant toxin B was greatly reduced, ascompared to the wild-type toxin B (e.g., SEQ ID NO: 2), which wasobtained commercially from List Biologicals), and as compared to thetriple mutant toxin B (SEQ ID NO: 86). The “cells only” control relatesto IMR-90 cells. See Table 62 below, showing the respective EC₅₀ values.

TABLE 62 Fold-reduction Sample EC₅₀ in EC₅₀ Penta Mutant B (lysateconcentrate) 11.5 μg/mL 1,642,857 Triple Mutant B (Purified) 18.9 ng/mL2700 List Toxin B lot127902-115  7.0 pg/mL 1

Moreover, the penta mutant toxin B (“PM-B”)(SEQ ID NO: 184) was assessedfor competitive inhibition of cytotoxicity that is mediated by thetriple mutant toxin B (SEQ ID NO: 86) on IMR-90 cells. See FIG. 27. Toprepare the samples, the triple mutant toxin B (TM B)(SEQ ID NO: 86) waskept at a constant concentration of 200 ng/mL (10×EC₅₀) in all wells. A2-fold serially diluted penta mutant toxin B starting at 5 μg/mL wasadded to the wells containing the triple mutant toxin B. The sampleswere then transferred to a 96-well plate containing IMR-90 cells, thenincubated for 72 hours. An ATP assay was completed, as described above,e.g., in Example 14. As can be shown in FIG. 27, the penta mutant toxinB competitively inhibited cytotoxicity of the triple mutant toxin B.

Example 16 Residual Cytotoxicity of Triple Mutant Toxins A and B (SEQ IDNOs: 4 and 6, Respectively)

To further evaluate the nature of the residual cytotoxicity, the mutanttoxins (SEQ ID NOs: 4 and 6) were mixed and incubated with theirrespective neutralizing antibodies before and the mixture was added toIMR90 cell monolayer.

The results (FIG. 8) showed that the residual cytotoxicity of mutanttoxin A and B (SEQ ID NOs: 4 and 6, respectively) can be completelyabrogated with neutralizing antibodies specific for mutant toxin A (toppanel, FIG. 8) and mutant toxin B (bottom panel, FIG. 8). Increasingconcentrations of mutant toxin A (top panel) and B (bottom panel) wereincubated with rabbit anti-toxin polyclonal (pAb, 1:10 dilution) ormurine monoclonal antibodies (1:50 dilution from a stock containing 3.0mg IgG/mL) before adding to IMR90 cells. After 72-hr treatmentincubation with IMR90 cells at 37° C., CellTiter-Glo® substrate wasadded and the relative light units (RLU) were measured in aspectrophotometer with the luminescence program to measure ATP levels.The lower the ATP level, the higher the toxicity. Controls included TcdAand TcdB added at 4 times their corresponding EC₅₀ values.

Published reports suggest that mutations in the glucosyltransferase orautocatalytic protease domain of the toxins result in completeinactivation of the toxicity. However, our data do not agree with thesepublished reports and this could be attributed to increasedconcentrations of the highly purified mutant toxins tested in ourstudies as opposed to crude culture lysates in published reports;increased time points at which cell rounding of mutant toxin-treatedcells was observed (e.g., 24 hours, 48 hours, 72 hours, or 96 hours) asopposed to observations made in less than 12 hours; use of cell linesthat exhibit significantly higher sensitivities to toxins in presentcytotoxicity assays in contrast to HT-29 human colorectal adenocarcinomacells in cytotoxicity assays disclosed in published reports; and/or toan unknown activity or process, other than glycosylation, that could bedriving the residual toxicity of the mutant toxins.

Example 17 Novel Mechanism of Cytotoxicity of Genetic Mutant Toxins

To investigate the mechanism of residual cytotoxicity of the geneticmutant toxins, IMR-90 cells were treated with wt toxin B (SEQ ID NO: 2)or genetic mutant toxin B (SEQ ID NO: 6), and glucosylation of Rac1GTPase was studied with time of treatment. Samples were collected from24 to 96 hours and cell extracts were prepared. Glucosylated Rac1 isdistinguished from non-glucosylated Rac1 by western blots with twoantibodies to Rac1. One antibody recognizes both forms of Rac1 (23A8)and the other (102) only recognizes non-glucosylated Rac1. Asillustrated in FIG. 22, for toxin B, the total Rac1 levels stayedunchanged over time with majority of the Rac1 being glucosylated.Treatment with the genetic mutant toxin B (SEQ ID NO: 6), on the otherhand, resulted in significant reduction of total Rac1, however, the Rac1was non-glucosylated at all time points. This shows that Rac1 level wasnegatively affected by the treatment with the genetic mutant toxin, butnot by wt toxin. As illustrated in FIG. 22, the level of actin wassimilar in toxin and genetic mutant toxin B treated cells and similar tomock treated cells at indicated time points. This showed that thegenetic mutant toxins exerted cytotoxicity by a mechanism which isdifferent than the wild-type toxin-driven glucosylation pathway.

Example 18 Chemical Treatment of Genetic Mutant Toxins

Although the genetically modified mutant toxins showed a 4-log reductionin cytotoxic activity is preferred, further reduction (2 to 4 logs) incytotoxic activity was considered. Two chemical inactivation strategieshave been evaluated.

The first method uses formaldehyde and glycine to inactivate the mutanttoxins. Formaldehyde inactivation occurs by forming a Schiff base(imine) between formaldehyde and primary amines on the protein. TheSchiff bases can then react with a number of amino acid residues (Arg,His, Trp, Tyr, Gln, Asn) to form either intra- or intermolecularcrosslinks. This crosslinking fixates the structure of the proteinrendering it inactive. In addition, formaldehyde can react with glycineto from a Schiff base. The glycyl Schiff base can then react with theamino acid residues to form intermolecular protein-glycine crosslinks.Formaldehyde reduced the cytotoxic activity of the genetic mutant toxinsto below detectable limits (reduction in cytotoxicity >8 log₁₀ fortriple mutant B (SEQ ID NO: 6) and >6 log₁₀ for triple mutant A (SEQ IDNO: 4). However, reversion was observed over time when theformaldehyde-inactivated (FI) triple mutant toxins were incubated at 25°C. The cytotoxic reversion can be prevented by addition of a low amountof formaldehyde (0.01-0.02%) into the FI-triple mutant toxins storagesolution. See Example 23.

Another method uses 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC)/N-hydroxysuccinimide (NHS) treatment to generate inactivatedmutant toxins. In this method, EDC/NHS reacts with carboxylic groups onthe protein to form activated esters. The activated esters can thenreact with primary amines on the protein to form stable amide bonds. Aswith the formaldehyde reaction, this reaction results in intra- andintermolecular crosslinks. The amide bond formed by treatment withEDC/NHS is more stable and non-reversible than the labile imine bondformed by formalin inactivation. In addition to crosslinks formed by thereaction of activated esters with primary amines on the polypeptide,both glycine and beta-alanine adducts can be formed. Without being boundby mechanism or theory, glycine adducts are produced when glycine isadded to quench unreacted activated esters. The amine of glycine reactswith the activated ester on the polypeptide to form stable amide bonds.Without being bound by mechanism or theory, beta-alanine adducts areformed by the reaction of activated beta-alanine with primary amines onthe polypeptide. This reaction results in stable amide bonds. Activatedbeta-alanine is produced by the reaction of three moles of NHS with onemole of EDC.

To achieve the 2-4 logs reduction of cytotoxic activity relative to thegenetically modified mutant toxins (6-8 logs, relative to nativetoxins), the chemically inactivated mutant toxins should have EC₅₀values of 000 μg/mL. In addition to reduction in cytotoxic activity, itwould be advantageous to retain key epitopes as determined by dot-blotanalysis. To date, a number of reaction conditions have been identifiedthat meet both the reduction cytotoxicity and epitope recognitioncriteria. Several batches of inactivated mutant toxins have beenprepared for animal studies and analytical data from a fewrepresentative batches is shown in Tables 7A and 7B, Table 8A and 8B.

TABLE 7A Chemically Inactivated Mutant Toxin A is Safe and Antigenic CPEReduction in EC₅₀ toxicity Reactivities to Sample # Toxin Sample IDTreatment μg/mL Log Scale mAbs 1 Mutant TcdA (SEQ ID NO: 4)Formalin >1000 6.4 Medium/high L44905-160A 2 Mutant TcdA (SEQ ID NO: 4)EDC >1000 6.4 High L44166-166 3 Mutant TcdA (SEQ ID NO: 4)Formalin >1000 6.4 Low L44905-170A CONTROLS 4 TcdA wt (from List Bio)none  390 pg/mL 1  High 5 TcdB wt (from List Bio) none 3.90 pg/mL Notapplicable None 6 rMutant TcdA TM Genetic none 12.5 μg/mL 4.5 HighL36901-79 (SEQ ID NO: 4) 7 Toxoid A List Bio Formalin Not Done — Low

TABLE 7B Chemically inactivated Mutant Toxin A is Safe and AntigenicReactivity with mAb (dot blot, nondenaturing conditions) Mid- N- DomainC-terminal (neut) Sample terminal mAb # A80- A3- A60- A65- # ToxinSample ID Treatment mAb #6 102 29 25 22 33 1 Mutant TcdA Formalin ++ ++++++ ++ ++++ ++++ (SEQ ID NO: 4) L44905-160A 2 Mutant TcdA EDC ++++ ++++++++ ++++ ++++ ++++ (SEQ ID NO: 4) L44166-166 3 Mutant TcdA Formalin + +++ ++ ++ + (SEQ ID NO: 4) L44905-170A CONTROLS 4 TcdA wt (from none +++++++ ++++ ++++ ++++ ++++ List Bio) 5 TcdB wt (from none — — — — — — ListBio) 6 rMutant TcdA none ++++ ++++ ++++ ++++ ++++ ++++ TM GeneticL36901-79 (SEQ ID NO: 4) 7 Toxoid A Formalin — — + — ++ + List BioList=List Biologicals; CPE=cytopathic effect assay; EC₅₀=the lowestconcentration where 50% of the cells show cytotoxicity; mAbs=monoclonalantibodies; neut=neutralizing; ND=not done; TM=active site and cleavagemutant (“triple mutant”)

TABLE 8A Chemically Inactivated Mutant Toxin B is Safe and Antigenic CPEReduction in EC₅₀ toxicity Reactivities to Sample # Toxin Sample IDTreatment μg/mL Log Scale mAbs 1 Mutant TcdB L44905-182 Formalin >10008.4 Medium/high (SEQ ID NO: 6) 2 Mutant TcdB L34346-38A EDC >1000 8.4High (SEQ ID NO: 6) 3 Mutant TcdB L44905-170B Formalin >1000 8.4 Low(SEQ ID NO: 6) CONTROLS 4 Tcda wt (from List Bio) none  390 pg/mL Notapplicable None 5 TcdB wt (from List Bio) none 3.90 pg/mL 1  High 6rMutant toxin B TM Genetic none   69 ng/mL 4.2 High (SEQ ID NO: 6)L34346-022 7 Toxoid A List Formalin Not done — Medium

TABLE 8B Chemically Inactivated Mutant Toxin B is Safe and AntigenicReactivity with mAb (dot blot, nondenaturing conditions) N-terminalMid-/C-terminal Sample aa 1-543 aa 544-2366 # Toxin Sample ID TreatmentB8-26 B9-30 B56-6 B59-3 1 Mutant TcdB (SEQ ID NO: 6) Formalin +++ +++ ++++ L44905-160A 2 Mutant TcdB (SEQ ID NO: 6) EDC ++++ ++++ ++++ ++++L44166-166 3 Mutant TcdB (SEQ ID NO: 6) Formalin ++ + +/− — L44905-170ACONTROLS 4 TcdA wt (from List Bio) none — — — — 5 TcdB wt (from ListBio) none ++++ +++ ++++ ++++ 6 rMutant TcdB TM Genetic none ++++ ++++++++ ++++ L34346-022 (SEQ ID NO: 6) 7 Toxoid B Formalin +++ +++ +++ +++ListList=List Biologicals; CPE=cytopathic effect assay; EC₅₀=theconcentration where 50% of the cells show cytotoxicity; mAbs=monoclonalantibodies; neut=neutralizing; ND=not done; TM=active site and cleavagemutant (“triple mutant”)

Example 19 Fermentation and Purification

Fermentations were initiated from a frozen source of a recombinantClostridium difficile including an fdx promoter described above. Thefrozen stocks were a cell suspension made to an OD₆₀₀=2.0 and 20%glycerol. A starter culture was inoculated with 0.2 mL of the culturestock into 500 mL SHYG10 medium (30 g/L soy hydrolysate SE50MK, 20 g/Lyeast extract HY YEST 412, 10 g/L glucose, 15 mg/L thiamphenicol, pH7).The medium was contained in a 500 mL vented bottle. The inoculation wasperformed in a conventional biosafety cabinet, the bottle was flushedwith nitrogen, and the bottle was then incubated static (vents closed)for ˜16-18 hours at 37° C. in a conventional incubator.

A ten liter bioreactor containing 8 L of SHYG60 medium (30 g/L soyhydrolysate SE50MK, 20 g/L yeast extract HY YEST 412, 60 g/L glucose, 15mg/L thiamphenicol, pH7) was used for the fermentation phase. The 500 mLcontents of the inoculum bottle were inoculated to the fermentor whichwas operated at 37° C., 400 rpm (1.47 m/s) with 0.1 vvm nitrogen sparge.The pH was controlled at 7.0 by auto-addition of 5N NaOH. Fermentationwas typically run for ˜24 hours to reach peak potency. Growth during thecourse of the fermentation was monitored by OD₆₀₀ readings. Samplestaken for mutant toxin quantitation were spun at ˜5000×g and theresulting pellets were frozen at −70° C. The cell pellet was thendefrosted and resuspended in a buffer consisting of 20 mM Tris, 3 mMNaCl, 0.5 mM EDTA, pH 6.5 and sonicated at an amplitude of 40 for 20seconds. The resulting cell lysate was spun at 5,000×g for 10 min. Thesupernatant was combined with loading buffer and reducing agent and runon a 3.8% Tris-acetate PAGE gel at 150 volts for 50-55 min versusauthentic toxin standards. The gel was stained overnight, then destainedand quantitated on a scanning densitometer. Typically, OD₆₀₀ values of˜10-12 were observed with toxin values of 80-120 mg/L.

The following table is an example of fermentation data for mutant toxinA.

TABLE 63 Elapsed fermentation Toxin yield time (hrs) OD₆₀₀ (mg/L) 1 0.323 1.16 5 3.55 33 7 5.56 53 9 7.11 63 11 8.64 81 24 11.14 123An example of fermentation data for mutant toxin B is presented in thetable below.

TABLE 64 Elapsed fermentation Toxin yield time (hrs) OD₆₀₀ (mg/L) 1 0.733 1.18 5 4.54 30 7 5.93 40 9 7.05 48 11 8.44 62 24 10.03 94

Modifications of the composition and methods for culturing therecombinant C. difficile cell and/or production of the mutant toxinshave been tested and are within the scope of the invention. For example,although SE50MK (and variously SE50MK-NK, both sourced fromFriesland-Campaigna) was the preferred choice for nitrogen source, avariety of other soy hydrolysates from other manufacturers wereidentified that worked well in the process. A culture medium includingsoy hydrolysate in the absence of yeast extract provided 30-40% of theexpected yield.

Yeast extracts from alternative manufacturers were shown to supportequivalent yields. A culture medium including yeast extract in theabsence of soy hydrolysate provided 60-70% of the expected yield.

Use of a culture medium based on soy hydrolysate/yeast extract, in theabsence of a carbon source, yielded about 2-3 OD₆₀₀ and about 10-15 mg/Ltoxin. Although glucose was a preferred carbon source for culturing,equivalent results were obtained with mannitol. A screen of carbonsources in bottles, indicated that C. difficile may also utilizefructose and mannose which would also be expected to support highOD/yield, as compared to a culture medium in the absence of a carbonsource. The following carbon sources did not appear to support optimalgrowth: arabinose, xylose, sucrose, lactose, maltose, glycerol, rhamnoseand galactose.

In addition, extending the fermentation time to 48 hours (requiringaddition of more glucose) did not appear to substantially improve yield.

Further, fermentation at pH 6.5 and 7.5 gave yields in the expectedrange of 80-120 mg/L. Fermentation at more extreme pH (pH 6.0 or 8.0)still gave the expected OD₆₀₀ values, but reduced yields (40-60 mg/L) oftoxin.

Fermentation using a culture medium in the absence of thiamphenicolresulted in a loss of plasmid, e.g., about 10-20% for a plasmid encodingmutant toxin B, and about 30-40% for a plasmid encoding mutant toxin A.Accordingly, fermentation using a culture medium in the absence of achloramphenicol derivative was feasible.

Alternative modes of operating fermentation were also tested and arewithin the scope of the invention. For example, fermentation may be runat 400 rpm or less and nitrogen overlay may be used, both techniques ofwhich were tried and used successfully.

Moreover, a monoclonal antibody medium SFM4MAb was tested and was shownto give about 10 OD₆₀₀ of cells and about 40 mg/L of mutant toxin.

Lastly, addition of a phosphate-containing ingredient to thefermentation appeared to reduce the production of toxin, as compared toculture medium in the absence of the phosphate-containing ingredient.

At the end of fermentation, the fermenter is cooled. The cell slurry isrecovered by continuous centrifugation and re-suspended in theappropriate buffer. Lysis of the cell suspension is achieved byhigh-pressure homogenization. For mutant toxin A, the homogenate isflocculated and the flocculated solution undergoes continuouscentrifugation. This solution is filtered and then transferred fordownstream processing. For mutant toxin B, the homogenate is clarifiedby continuous centrifugation, and then transferred for downstreamprocessing.

Mutant toxin A (SEQ ID NO: 4) is purified using two chromatographicsteps followed by a final buffer exchange. The clarified lysate isloaded onto a hydrophobic interaction chromatography (HIC) column andthe bound mutant toxin is eluted using a sodium citrate gradient. Theproduct pool from the HIC column is then loaded on a cation exchange(CEX) column and the bound mutant toxin A is eluted using a sodiumchloride gradient. The CEX pool containing purified mutant toxin A isexchanged into the final buffer by diafiltration. The purified mutanttoxin A is exchanged into the final drug substance intermediate bufferby diafiltration. After diafiltration, the retentate is filtered througha 0.2 micron filter prior to chemically inactivation to a final drugsubstance. The protein concentration is targeted to 1-3 mg/mL.

Mutant toxin B (SEQ ID NO: 6) is purified using two chromatographicsteps followed by a final buffer exchange. The clarified lysate isloaded onto an anion exchange (AEX) column, and the bound mutant toxinis eluted using a sodium chloride gradient. Sodium citrate is added tothe product pool from the AEX column and loaded on a hydrophobicinteraction chromatography (HIC) column. The bound mutant toxin iseluted using a sodium citrate gradient. The HIC pool containing purifiedmutant toxin polypeptide (SEQ ID NO: 6) is exchanged into the finalbuffer by diafiltration. The purified mutant toxin B is exchanged intothe final drug substance intermediate buffer by diafiltration. Afterdiafiltration, the retentate is filtered through a 0.2 micron filterprior to chemically inactivation to a final drug substance. The proteinconcentration is targeted to 1-3 mg/mL.

Example 20 Formaldehyde/Glycine Inactivation

After purification, the genetic mutant toxins A and B (SEQ ID NOs: 4 and6, respectively) are inactivated for 48 hours at 25° C. using 40 mM (1.2mg/ml) of formaldehyde. The inactivation is carried out at pH 7.0±0.5 in10 mM phosphate, 150 mM sodium chloride buffer containing 40 mM (3mg/ml) glycine. The inactivation period is set to exceed three times theperiod needed for reduction in the EC₅₀ in IMR90 cells to greater than1000 ug/mL. After 48 hours, the biological activity is reduced 7 to 8log₁₀ relative to the native toxin. Following the 48 hour incubation,the inactivated mutant toxin is exchanged into the final drug substancebuffer by diafiltration. For example, using a 100 kD regeneratedcellulose acetate ultrafiltration cassette, the inactivated toxin isconcentrated to 1-2 mg/mL and buffer-exchanged.

Example 21 N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide(EDC)/N-hydroxysuccinimide (NHS) Inactivation

After purification, the genetic mutant toxins (SEQ ID NO: 4 and SEQ IDNO: 6) are inactivated for 2 hours at 25° C. using 0.5 mg EDC and 0.5 mgNHS per mg of purified genetic mutant toxin A and B (approximately 2.6mM and 4.4 mM respectively). The reaction is quenched by the addition ofglycine to a final concentration of 100 mM and the reactions incubatefor an additional 2 hours at 25° C. The inactivation is carried out atpH 7.0±0.5 in 10 mM phosphate, 150 mM sodium chloride buffer. Theinactivation period is set to exceed three times the period needed forreduction in the EC₅₀ in IMR90 cells to greater than 1000 ug/mL. After 2hours, the biological activity is reduced 7 to 8 log₁₀ relative to thenative toxin. Following the 4 hour incubation, the inactivated mutanttoxin is exchanged into the final drug substance buffer bydiafiltration. For example, using a 100 kD regenerated cellulose acetateultrafiltration cassette, the inactivated toxin is concentrated to 1-2mg/mL and buffer-exchanged.

Unless otherwise stated, the following terms as used in the Examplessection refer to a composition produced according to the presentdescription in Example 21: “EDC/NHS-treated triple mutant toxin”;“EDC-inactivated mutant toxin”; “mutant toxin [NB] drug substance”;“EI-mutant toxin”; “EDC/NHS-triple mutant toxin.” For example, thefollowing terms are synonymous: “EDC/NHS-treated triple mutant toxin A”;“EDC-inactivated mutant toxin A”; “mutant toxin A drug substance”;“EI-mutant toxin A”; “EDC/NHS-triple mutant toxin A.” As anotherexample, the following terms are synonymous: “EDC/NHS-treated triplemutant toxin B”; “EDC-inactivated mutant toxin B”; “mutant toxin B drugsubstance”; “EI-mutant toxin B”; “EDC/NHS-triple mutant toxin B.”

The mutant toxin A drug substance and the mutant toxin B drug substanceare each manufactured using a batch process, which includes (1)fermentation of a the toxin negative C. difficile strain (VPI 11186)containing a plasmid encoding the respective genetic triple mutant toxinpolypeptide (in a medium including soy hydrolysate, yeast extract HYYEST™ 412 (Sheffield Bioscience), glucose, and thiamphenicol), (2)purification of the genetic mutant toxin (the “drug substanceintermediate”) from the cell-free lysate using ion exchange andhydrophobic interaction chromatographic procedures to at least greaterthan 95% purity, (3) chemical inactivation by treatment with EDC/NHSfollowed by quenching/capping with glycine, and (4) exchange into thefinal buffer matrix.

Example 22 Studies Supporting Conditions of Inactivation and Formulation

To optimize the chemical inactivation of the genetic mutant toxins, astatistical design of experiment (DOE) was performed. Factors examinedin the DOE included temperature, formaldehyde/glycine concentration,EDC/NHS concentration and time (Table 9 and 10). To monitor loss ofbiological activity, EC₅₀ values in IMR90 cells were determined. Inaddition, cell morphology of IMR-90 cells various timepointspost-treatment were also observed. See FIG. 9, showing morphology at 72hours post treatment. To determine the effect on protein structure,epitope recognition was monitored using dot-blot analysis using a panelof monoclonal antibodies raised against different domains of the toxin.

TABLE 9 Parameters Tested Formaldehyde/Glycine DOE Parameters Rangetested Time (days) 1 to 14 Temperature (° C.) 4 to 37 Toxinconcentration (mg/ml) 1 to 1.25 Formaldehyde concentration (mM) 2 to 80Glycine concentration (mg/ml) 0 to 80

TABLE 10 Parameters Tested EDC/NHS DOE Parameters Range tested Time(hours) 1 to 4 Temperature (° C.) 25 to 35 Toxin concentration (mg/ml) 1to 1.25 EDC (mg/mg toxin) 0.25 to 2.5 NHS (mg/mg toxin) 0 to 2.5

In the formaldehyde/glycine inactivation of C. difficile mutant toxins,final reaction conditions were chosen such that the desired level ofreduction in cytotoxic activity (7 to 8 log₁₀) was achieved whilemaximizing epitope recognition. See Example 20 above.

In the EDC/NHS inactivation of C. difficile mutant toxins, finalreaction conditions were chosen such that the desired level of reductionin cytotoxic activity (7 to 8 log₁₀) was achieved while maximizingepitope recognition. See Example 21 above.

In an alternative embodiment, the EDC-NHS reaction was quenched byaddition of alanine, which sufficiently quenched the reaction. Use ofalanine may result in a modification on the mutant toxin protein that issimilar to the modification when the reaction is quenched by glycine.For example, quenching by adding alanine may result in an alanine moietyon a side chain of a glutamic acid and/or aspartic acid residue of themutant toxin. In another alternative embodiment, the EDC-NHS reactionwas quenched by addition of glycine methyl ester, which sufficientlyquenched the reaction.

Production of chemically inactive triple mutant C. difficile toxin A andtoxin B under optimized conditions resulted in a further reduction ofresidual cytotoxicity to an undetectable level (>1000 μg/mL—the highestconcentration tested via the CPE assay), while retaining antigenicity asmeasured by their reactivity to the toxin-specific neutralizingantibodies. The results shown in Table 28 demonstrate a stepwisereduction in cytotoxicity from wt toxin through to EDC/NHS-treatedtriple mutant toxins. Immunofluorescence labelling confirmed that triplemutant toxins (SEQ ID NO: 4 and 6) and mutant toxin drug substancesexhibited comparable binding to the IMR-90 cells suggesting that thecytotoxicity loss was not due to reduced binding to the cells (data notshown). Compared to mutant toxin A drug substance, the mutant toxin Bdrug substance achieved higher fold-reduction in cytotoxicity, which isconsistent with the observed ˜600-fold higher potency of TcdB comparedto TcdA.

TABLE 28 Cytotoxicity Summary Fold reduction in Toxin Sample EC₅₀cytotoxicity A TcdA (SEQ ID NO: 1) 1.6 ng/mL 1 Triple mutant 12.5 μg/mL7800 toxin A (SEQ ID NO: 4) Mutant toxin A >1000 μg/mL >625,000 DrugSubstance B TcdB (SEQ ID NO: 2) 2.5 pg/mL 1 Triple mutant 45 ng/mL18,000 toxin B (SEQ ID NO: 6) Mutant toxin B >1000 μg/mL >400,000,000Drug SubstanceCytotoxicity assay results for mutant toxin B modified by EDC alone, orby EDC and sulfo-NHS were also assessed. See Table 29.

TABLE 29 Cytotoxicity EC₅₀, mg · Sample mL⁻¹ (CPE) Comment TcdB TM (SEQID NO: 6), 0.03 unmodified TM TcdB-EDC 1, no NHS <0.97 Reacted with EDCalone TM TcdB-EDC 2, no NHS <0.97 Duplicate preparation TM TcdB-EDC 3,sulfo- 125 Reacted with EDC and NHS (0.5x) sulfo-NHS TM TcdB-EDC 4,sulfo- 125 Duplicate preparation NHS (0.5x) TM TcdB-EDC 3, sulfo- 250Reacted with EDC and NHS (1.0x) sulfo-NHS TM TcdB-EDC 4, sulfo- 750Reacted with EDC and NHS (2.0x) sulfo-NHS

Conditions: Triple mutant toxin B (“TM TcdB”)(SEQ ID NO: 6) was modifiedin the weight ratios mutant toxin B:EDC:sulfo-NHS=1:0.5:0.94. This ratiois the molar equivalent (corrected for higher MW of sulfo-NHS) to thestandard EDC/NHS reaction as described in Example 21. To determine theaffect of sulfo-NHS, the sulfo-NHS ratio was varied from 0.5× to 2× thestandard ratio. Duplicate reactions were performed in 1×PBS pH 7.0 at25° C., and were initiated by addition of EDC solution. After 2 hours,reactions were quenched by the addition of 1 M glycine pH 7.0 (0.1 Mfinal concentration) and incubated for a further 2 hours. Quenchedreactions were desalted and mutant toxin B drug substance (“TMTcdB-EDC”) was concentrated using Vivaspin 20 devices, and sterilefiltered into sterile vials and submitted for assessment in acytotoxicity assay.

At the same molar ratio, sulfo-NHS reduced the EC₅₀ to about 250 ug/mLas compared to >1000 ug/mL for NHS. Even at twice the molar ratio,sulfo-NHS does not appear not as effective as NHS in decreasingcytotoxicity. See Table 30.

TABLE 30 reference NHS control Sulfo-NHS Digest (TcdB digest (TcdBSample Modification EDC 004) EDC 001) Digest glycine adduct (+57 da) 4929 35 beta-alanine (+71 da) 24 19 0 crosslinks (−18 da) 7 4 3dehydroalanine (−34 da) 6 5 4 Unmodified 273 195 217

To determine the number and type of modifications, peptide mapping wasperformed on both EDC/NHS and EDC/sulfo-NHS inactivated triple mutanttoxin B samples. Similar amounts of glycine adducts, crosslinks anddehydroalanine modifications were observed in both samples. However inthe sulfo-NHS sample, no beta-alanine was observed.

Wild-type toxin B (SEQ ID NO: 2) was inactivated using the standardprotocol (see Example 21); toxin B:EDC:NHS 1:0.5:0.5, 25° C. for 2 hoursin 1×PBS pH 7.0, then quench with 1 M glycine (0.1 M finalconcentration) and incubate for an additional 2 hours. The sample wasdesalted, concentrated and submitted for cytotoxicity assay. The EC₅₀for this samples was <244 ng/mL.

Example 23 Reversion Studies

To determine if reversion occurs with either the formaldehyde/glycine orEDC/NHS inactivated C. difficile mutant toxins, samples of inactivatedmutant toxins (1 mg/mL) were incubated at 25° C. for five-six weeks.Aliquots were removed each week and the EC₅₀ values in IMR90 cells weredetermined. One formaldehyde/glycine inactivated sample contained noformaldehyde and one sample contained 0.01% formaldehyde. The EC₅₀ wasmeasured by the CPE assay.

TABLE 11 Results from Inactivated TcdA Reversion Study EC₅₀ (IMR90 cellassay) Formalin-inactivated Time of Incubation No 0.01% (Days)formaldehyde formaldehyde EDC/NHS 0 1000 ug/ml  1000 ug/ml 1000 ug/ml 7740 ug/mL ND 1000 ug/ml 14 493 ug/mL 1000 ug/ml 1000 ug/ml 21 395 ug/mLND 1000 ug/ml 28 395 ug/mL 1000 ug/ml 1000 ug/ml 35 326 ug/M  ND ND

At 25° C. in the absence of residual formaldehyde, partial reversion isobserved (Table 11). After five weeks, the cytotoxic activity increasedapproximately 3-fold. Although the cytotoxic activity increased, afterfive weeks there was still a 7 log₁₀ reduction relative to the nativetoxin. Reversion was completely prevented by inclusion of formalin at aconcentration of 0.010%. No reversion was observed in the EDC/NHSinactivated sample. Throughout the 6-week incubation, EC₅₀ valuesremained at the starting level of >1000 μg/mL for all four lots of bothEDC/NHS-treated triple mutant toxin A (SEQ ID NO: 4) and EDC/NHS-treatedtriple mutant toxin B (SEQ ID NO: 6). In contrast, the EC₅₀ values ofFI-treated triple mutant toxin A (SEQ ID NO: 4) and FI-treated triplemutant toxin B (SEQ ID NO: 6) were not stable and declined tounacceptably low EC₅₀ values, indicating an increase in cytotoxicity orreversion of inactivation. See Table 11.

In addition to stably reducing the cytotoxicity to an undetectable level(>1000 μg/mL, as measured by the CPE assay), mutant toxins inactivatedusing EDC/NHS retained important epitopes that are targets oftoxin-neutralizing mAbs. See Table 31. FI mutant toxins showed a loss ofthe same antigenic determinants.

TABLE 31 EDC/NHS Inactivation Reduced Cytotoxicity of Genetic MutantToxins and Maintained Important Antigenic Determinants Reduction incytotoxicity Max binding (Rmax)^(b) relative to wt Neut mAb^(d) SampleEC₅₀ toxin (log₁₀)^(a) 1^(c) 2 3 Triple mutant A  12.5 μg/mL 4.5 100 100100 (SEQ ID NO: 4) FI- Triple >1000 μg/mL >6.4 55 59 53 mutant AEDC/NHS- Triple >1000 μg/mL >6.4 90 94 103 mutant A Triple mutant B   69ng/mL 4.3 100 100 100 (SEQ ID NO: 6) FI-Triple >1000 μg/mL 8.4 67 67 36mutant B EDC/NHS-Triple >1000 μg/mL 8.4 87 78 73 mutant B^(a)cytotoxicity was measured using the CPE assay on IMR90 cells^(b)values determined by Biacore ™ analysis using multiple neutralizingmAbs directed at various non-overlapping toxin epitopes ^(c)values areaverages of two experiments ^(d)For the first three rows, the neut mAb“1,” “2,” “3” refer to mAbs A60-22, A80-29, and A65-33 for toxin A,respectively. For the bottom three rows, the neut mAb “1,” “2,” “3”refer to mAbs B8-26, B59-3, and B-56-15 for toxin B, respectively.

Example 24 Preclinical Immunogenicity Studies

Key preclinical objectives include testing compositions including C.difficile mutant toxins A and B in small animals and nonhuman primates(NHP). Mice and hamsters were immunized to determine, among otherthings, if the C. difficile compositions are capable of elicitingneutralizing antibodies against the mutant toxin A and B. The antigenswere tested for induction of serum neutralization antibody responsesfollowing a series of immunizations in mice, hamsters, and cynomolgusmacaques. The genetic and/or chemically-inactivated mutant toxins wereformulated in either neutral buffer, aluminum phosphate buffer, orbuffer containing ISCOMATRIX as an adjuvant in some embodiments.Neutralizing antibody responses were generally tested about two to fourweeks after each boost or the final dose.

The toxin neutralization assay demonstrates the ability of an antiserumto neutralize the cytotoxic effect mediated by C. difficile TcdA or TcdBand is therefore able to measure the functional activity of antibodiesthat are present in a sample. A toxin neutralization assay was performedon a human lung fibroblast cell line, IMR-90, which is sensitive to bothTcdA and TcdB. Briefly, a 96-well microtiter plate was seeded withIMR-90 cells serving as the target of toxin-mediated cytotoxicity. Eachtest serum sample was analyzed separately for the ability to neutralizeTcdA and TcdB. Appropriate serial dilutions of test antisera were mixedwith a fixed concentrations of TcdA or TcdB and incubated at 37° C. for90 minutes in a humidified incubator (37° C./5% CO₂) to allow forneutralization of the toxins to occur. For quality control, all platesincluded a Reference standard and controls which includes antitoxinantibodies of known titer. After 90 minutes, the toxin-antisera mixturewas added to the IMR-90 cell monolayer and the plates were incubated foran additional 72 hours. Subsequently, CellTiter-Glo® substrate was addedto the assay plate to determine the Adenosine Triphosphate (ATP) levelspresent in metabolically active cells and was measured as RelativeLuminescence Units (RLU). A large ATP level indicates high cellviability, and levels are directly proportional to the amount ofneutralization of the toxin by the antibody present in the sample. Forpreclinical data, the RLU data was plotted against the dilution value ofthe test antisera sample to generate a Four-Parameter Logistic (4-PL)regression response fit curve. The neutralization titers were expressedas the sample dilution value which exhibited 50% reduction incytotoxicity.

Example 25 Mouse Immunogenicity Study: Mu C. difficile 2010-06

The purpose of this study was to assess the immunogenicity of two formsof mutant C. difficile toxin B (SEQ ID NO: 6), eachchemically-inactivated by different methods. In this study, theuntreated mutant toxin B (SEQ ID NO: 6) (genetically inactivated but notchemically inactivated) was used as a control, with and withoutadjuvant.

Groups of 10 mice were immunized intramuscularly with 10 μg of animmunogen according to Table 12.

TABLE 12 Testing chemically inactivated mutant toxin B (SEQ ID NO: 6) inmice Group Immunogen Dose No. Route Schedule 1 Formalin-Inactivated 10μg 10 IM Prime wk 0, Mutant toxin B^(a) Boost wks 4, 8 in AlPO₄ ^(c) 2Inactivated Mutant 10 μg 10 IM Prime wk 0, toxin B form 2^(b) Boost wks4, 8 in AlPO₄ ^(c) 3 Genetic-Inactivated 10 μg 10 IM Prime wk 0, Mutanttoxin B Boost wks 4, 8, unadjuvanted 4 Genetic-Inactivated 10 μg 10 IMPrime wk 0, Mutant toxin B Boost wks 4, 8, in AlPO₄ ^(c) ^(a)chemicalinactivation = Formalin/glycine treated 10° C. for 7 days ^(b)chemicalinactivation = EDC/NHS treated, 30° C. for 2 hours ^(c) aluminum ionconcentration = 0.5 mg/mLResults: There were no adverse events in the mice following eachadministration of the vaccine candidates. As illustrated in FIG. 10,mice in each group developed significant robust anti-toxin Bneutralizing antibodies after the third dose with the respective mutanttoxins.

Based on the week 12 titers, it appears that in mice the EDC-inactivatedmutant toxin B (Group 2) and the formalin-inactivated mutant toxins(Group 1) generated potent neutralizing responses.

In the absence of chemical inactivation, the genetic mutant toxin B (SEQID NO: 6) generated neutralizing responses after two doses (Groups 3-4,week 8), which were boosted after the third dose (Groups 3-4, week 12).

Example 26 Mouse Immunogenicity Study: Mu C. Difficile 2010-07

The purpose of this study was to assess immunogenicity of chemicallyinactivated C. difficile mutant toxins A and B (SEQ ID NOs: 4 and 6,respectively), either alone or in combination. The immunogens for allgroups were formulated with aluminum phosphate as an adjuvant.

Groups of 5 mice were immunized intramuscularly with 10 μg of animmunogen according to Table 13.

TABLE 13 Testing Chemically Inactivated Genetic A and B mutant toxins(SEQ ID NOs: 4 and 6, respectively) in Mice Group Immunogen Dose No.Group Schedule 1 Formalin-Inactivated^(a) 10 μg 5 IM Prime wk 0, Mutanttoxin B Boost wks 4, 8, (SEQ ID NO: 6) 12 in AlPO₄ ^(c) 2EDC-Inactivated^(b) 10 μg 5 IM Prime wk 0, Mutant toxin B Boost wks 4,8, (SEQ ID NO: 6) 12 in AlPO₄ ^(c) 3 Formalin-Inactivated 10 μg 5 IMPrime wk 0, Mutant toxin A Boost wks 4, 8, (SEQ ID NO: 4) 12 form 1 inAlPO₄ ^(c) 4 EDC-Inactivated 10 μg 5 IM Prime wk 0, Mutant toxin A Boostwks 4, 8, (SEQ ID NO: 4) 12 in AlPO₄ ^(c) 5 Formalin-Inactivated 10 μg 5IM Prime wk 0, Mutant toxins A + B each Boost wks 4, 8, in AlPO₄ ^(c) 12^(a)Formalin-treatment = formalin/glycine treated for 2 days at 25° C.;mutant toxin was not cytotoxic and retained binding to all mutanttoxin-specific monoclonal antibodies tested ^(b)EDC-treatment = EDC/NHStreated for 4 hrs at 30° C.; mutant toxin was not cytotoxic and retainedbinding to all mutant toxin-specific monoclonal antibodies tested^(c)aluminum ion concentration = 0.5 mg/mL

Results: There were no adverse events in the mice following eachadministration of the vaccine candidates. As illustrated in FIG. 11,after two doses of chemically inactivated genetic mutant toxins, theanti-toxin A neutralizing antibodies (Groups 3-5) were boosted to titersbetween 3 and 4 log₁₀ while the anti-toxin B neutralizing antibodies(Groups 1-2, 5) remained low to undetectable, which is consistent withthe data from the mouse study described above (FIG. 10). Anti-toxin Bneutralizing antibodies boosted to 2-3 log₁₀ in groups 1, 2, and 5following the third dose (week 12 titers) and reached their peak twoweeks following the fourth dose (week 14 titers). The anti-toxin Aneutralizing antibody titers in groups 3-5 increased slightly followingthe third (week 12 titers) and fourth immunizations (week 14 titers).

Example 27 Hamster Immunogenicity Study: Ham C. Difficile 2010-02

The purpose of this study was to assess immunogenicity and protectivepotential of C. difficile triple mutant and chemically inactivatedmutant toxins A and B in the Syrian golden hamster model. The Syriangolden hamster model represents the best available challenge model forsimulating human CDAD. The same batches of mutant toxins A and B used inmouse study muC. difficile 2010-07 were used in this study. As acontrol, one group was given mutant toxins without aluminum-containingadjuvant.

Groups of 5 Syrian golden hamsters were immunized intramuscularly with10 μg of an immunogen according to Table 14.

TABLE 14 Testing Chemically Inactivated Mutant Toxins A and B (SEQ IDNOs: 4 and 6, respectively) in Hamsters (hamC. difficile2010-02) GroupImmunogen Dose No. Route Schedule 1 Formalin-Inactivated^(a) 10 μg 5 IMPrime wk 0, Mutant toxins A + B each Boost wks 4, 8, (SEQ ID NOs: 4 and6) 12 in AlPO₄ ^(c) 2 Formalin-Inactivated 10 μg 5 IM Prime wk 0, Mutanttoxins A + B each Boost wks 4, 8, (SEQ ID NOs: 4 and 6) 12 in PBS (noadjuvant) 3 EDC-Inactivated^(b) 10 μg 5 IM Prime wk 0, Mutant toxins A +B each Boost wks 4, 8, (SEQ ID NOs: 4 and 6) 12 in AlPO₄ ^(c) 4 ListBiological toxoid 10 μg 5 IM Prime wk 0, in AlPO₄ ^(c) each Boost wks 4,8, 12 ^(a)Formalin-treatment = formalin/glycine treated for 2 days at25° C.; Mutant toxin was not cytotoxic and retained binding to allmutant toxin-specific monoclonal antibodies tested ^(b)EDC-treatment =EDC/NHS treated for 4 hrs at 30° C.; Mutant toxin was not cytotoxic andretained binding to all mutant toxin-specific monoclonal antibodiestested ^(c)aluminum ion concentration = 0.5 mg/mL 1. Animals: 15 Syriangolden hamsters, female, 6-8 weeks old/100-130 g each. 2. Vaccination:IM, 0.05 ml each, according to above schedule. Toxoids will be providedby Process Development and will be formulated in AlPO4 diluent by theFormulations Group. Group 2 will serve as a non-adjuvanted controlgroup. 3. Bleed: All hamsters will be bled at weeks 0, 4, 8, and 12,just prior to each immunization. 4. Serum sample analysis:Neutralization assay

Results: There were no adverse events observed following immunizationwith the mutant toxins. As illustrated in FIG. 12, after a single doseof mutant toxins, the anti-toxin A neutralizing responses were between2-3 log₁₀ for the formalin-inactivated mutant toxins (Groups 1-2) andbetween 3-4 log₁₀ for the EDC-inactivated mutant toxins (Group 3). Afterthe second dose, anti-toxin A antibodies boosted in all three groups.Anti-toxin A antibodies in all three groups did not appear to increaseafter the third dose. A similar result was observed after the fourthimmunization, where an increase in titer was observed in theformalin-inactivated group that did not contain the aluminum adjuvant(Group 2).

The anti-toxin B neutralizing responses were undetectable in theformalin-inactivated mutant toxins groups (Groups 1-2) and were justover 2 log₁₀ for the EDC-inactivated mutant toxins (Group 3) after asingle dose. After the second dose, anti-toxin B neutralizing antibodytiters in the two formalin-inactivated groups (Groups 1-2) increased to3-4 log₁₀ while those in the EDC-inactivated group (Group 3) increasedto 4-5 log₁₀. For all three groups, increases in anti-toxin Bneutralizing antibody titers were observed after the third and/or fourthdoses, with all groups reaching a peak titer at week 16 (after the lastdose). See FIG. 12.

In FIG. 13, the level of neutralizing antibody responses againstchemically inactivated genetic mutant toxins (FIG. 12) was compared tothose elicited by List Biological Laboratories, Inc. (Campbell, Calif.)(also referred herein as “List Bio” or “List Biologicals”) toxoids(i.e., toxoids purchased from List Biological Laboratories were preparedby formalin inactivation of wild type toxins; control reagent used toestablish the hamster challenge model).

As used herein, “FI” in figures and tables refers to formalin/glycinetreatment of the toxins, 2 days at 25° C., unless otherwise stated. Asused herein, “EI” in figures and tables refers to EDC/NHS treatment for4 hours at 30° C., unless otherwise stated. In FIG. 13, 5 hamsteranimals were treated with the respective mutant toxin composition,whereas 11 hamster animals were treated with the toxoid purchased fromList Biological.

The data in FIG. 13 shows that, in hamsters administered according toTable 14, the respective neutralizing antibody titers against toxin A(FIG. 13A) and toxin B (FIG. 13B) induced by the immunogenic compositionincluding EDC inactivated mutant toxins after two doses is higher thanthe respective neutralizing antibody titers elicited by the ListBiologicals toxoids.

Example 28 Hamster Immunogenicity Study: C. difficile Ham2010-02(Continued)

To assess protective efficacy of the mutant toxins, immunized hamsters,along with one control group of non-immunized animals, were first givenan oral dose of clindamycin antibiotic (30 mg/kg) to disrupt normalintestinal flora. After five days, the hamsters were challenged with anoral dose of wild type C. difficile spores (630 strain, 100 cfu peranimal). Animals were monitored daily for eleven days post-challenge forsigns of CDAD, which in hamsters is known as wet tail. Using a system ofclinical scoring a number of different parameters, animals determined tohave severe CDAD were euthanized. The parameters included activityfollowing stimulation, dehydration, excrement, temperature, and weight,etc., which are known in the art.

At day 11, the study was terminated and all surviving animals wereeuthanized. FIG. 14 shows the survival curves for each of the threeimmunized groups (Groups 1-3, according to Table 14) as compared to thenon-immunized controls. As can be seen, the non-immunized animals alldeveloped severe CDAD and required euthanasia between days 1-3 postchallenge (0% survival). Both groups administered withformalin-inactivated mutant toxin had 60% survival curves, with animalsnot requiring euthanasia until day 3 (Group 1) or day 4 (Group 2). Thegroup administered with EDC-inactivated mutant toxin had an 80% survivalcurve, with 1 (out of 5) animal requiring euthanasia on day 7.Accordingly, the hamsters were protected from lethal challenge with C.difficile spores.

Example 29 Hamster Immunogenicity Study: Ham C. difficile 2010-03:Immunogenicity of Genetic and Chemically-Inactivated C. difficile MutantToxins

The purpose of this study was to assess immunogenicity of non-adjuvantedC. difficile triple mutant and chemically inactivated mutant toxins Aand B (SEQ ID NOs: 4 and 6, respectively) in the Syrian golden hamstermodel. The same batches of mutant toxins A and B (SEQ ID NOs: 4 and 6,respectively) used in mouse study mu C. difficile 2010-07 were used inthis study. As a control, one group (Group 1) was given aphosphate-buffered saline as placebo.

Groups of five or ten Syrian golden hamsters were immunized with animmunogen according to Table 15. Animals were given three doses. Inaddition, animals were dosed every two weeks.

TABLE 15 Experimental Design of Hamster Immunization and Challenge GroupImmunogen Dose No. Route Schedule 1 Placebo (PBS buffer) NA 5 NA 2Mutant toxin A + B 10 μg 10 IM Prime wk 0, (SEQ ID NOs: 4 and each Boostwks 2, 4 6, respectively); Formalin-inactivated 3 Mutant toxin A + B 10μg 10 IM Prime wk 0, (SEQ ID NOs: 4 and each Boost wks 2, 4 6,respectively); EDC- Inactivated 4 Mutant toxin A + B 10 μg 10 IM Primewk 0, (SEQ ID NOs: 4 and each Boost wks 2, 4 6, respectively); genetic

Results: See FIG. 15. No anti-toxin A or B antibodies were observed inthe placebo control group. After one dose, anti-toxin A neutralizingantibodies were observed between 2-3 log₁₀ for the formalin-inactivated(Group 2) and genetic mutant toxin (Group 4) groups and between 3-4log₁₀ for the EDC-inactivated group (Group 3). Anti-toxin A neutralizingantibodies increased in each of these groups (2-4) after the secondimmunization with the relevant mutant toxins (compare titers at week 2to week 3 in FIG. 15). After the third dose of mutant toxins (given atweek 4), anti-toxin A neutralizing antibody titers in Groups 2-4increased compared to their week 4 titers.

Anti-toxin B neutralizing antibodies were detectable after the seconddose, wherein the formalin-inactivated (Group 2) and EDC-inactivated(Group 3) anti-toxin B neutralizing antibodies increased to between 3-4log₁₀ and to between 2-3 log₁₀ for the genetic triple mutant (Group 4).Following the third immunization (week 4), the anti-toxin B neutralizingantibody titers boosted to between 3-4 log₁₀ for theformalin-inactivated mutant toxins (Group 2) and genetic mutant toxins(Group 4) and between 4-5 log₁₀ for the EDC-inactivated mutant toxins(Group 3).

For both anti-toxin A and anti-toxin B neutralizing antibodies, peaktiters were observed at week 6 (post-dose 3) for all vaccinated groups(Groups 2-4).

Assessment of Immunogenic Compositions Adjuvanted with Alhydrogel/CpG orISCOMATRIX

Hamsters immunized with an immunogenic composition including achemically inactivated mutant toxin formulated with Alhydrogel,ISCOMATRIX, or Alhydrogel/CpG24555 (Alh/CpG) developed robustneutralizing antitoxin antisera. It was observed that peak antitoxin Aand antitoxin B responses were 2-3-fold higher and statisticallysignificant in groups immunized with mutant toxins formulated in Alh/CpGor ISCOMATRIX when compared to vaccine formulated with Alhydrogel alone.See Table 32 showing 50% neutralization titers. Hamsters (n=10/group)were immunized IM at 0, 2, and 4 weeks with 10 μg each mutant toxin Adrug substance and mutant toxin B drug substance formulated with 100 μgof Alhydrogel, or 200 μg of CpG 24555+100 μg of Alhydrogel, or 10 U ofISCOMATRIX. Sera were collected at each time point and analyzed in thetoxin neutralization assay for functional antitoxin activity. Geometricmean titers are provided in Table 32. Asterisks (*) indicate statisticalsignificance (p<0.05) when compared to titers in the Alhydrogel group.

TABLE 32 Immunogenicity of Adjuvanted Mutant Toxin Drug Substances inHamsters 50% Neutralization Titer Week 0 Week 1 Week 2 Week 3 Week 4Week 6 Antitoxin A Alhydrogel 10 26 88 7425 6128 15965 Titer: Alh/CpG 10103 *688 *34572 *23028 *62203 Titer: ISCOMATRIX 10 27 *246 *12375 8566*36244 Titer: Antitoxin B Alhydrogel 10 15 10 218 1964 7703 Titer:Alh/CpG 10 10 18 *5550 *5212 *59232 Titer: ISCOMATRIX 10 12 12 *7412*15311 *92927 Titer:

Protective efficacy of the immunogenic composition including mutanttoxin drug substances formulated with these adjuvants was tested.Hamsters were immunized and were given oral clindamycin (30 mg/kg) onweek 5 and challenged according to the method described above. One groupof unimmunized hamsters (n=5) was included as a control. Increasedefficacy was observed in hamsters immunized with mutant toxin drugsubstances adjuvanted with either Alh/CpG or ISCOMATRIX (100% survival)as compared to Alhydrogel alone (70% survival). Accordingly, thehamsters were protected from lethal challenge with C. difficile spores.

Example 30 Clostridium difficile Vaccination in Cynomolgus Macaques

The purpose of this study was to test the immunogenicity of low and highdoses of EDC-Inactivated and Formalin-Inactivated C. difficile mutanttoxins in cynomolgus macaques. All mutant toxins were formulated inISCOMATRIX® as an adjuvant except for one group, which served as theunadjuvanted control (Group 5).

TABLE 16 Immunization of Cynomolgus Macaques Group Immunogen Number DoseRoute Schedule 1 FI-Mutant toxins 5 10 μg IM Prime wk 0, A + B eachBoost wks 2, 4 (ISCOMATRIX) 2 FI-Mutant toxins 5 100 μg IM Prime wk 0,A + B each Boost wks 2, 4 (ISCOMATRIX) 3 EI-Mutant toxins 5 10 μg IMPrime wk 0, A + B each Boost wks 2, 4 (ISCOMATRIX) 4 EI-Mutant toxins 5100 μg IM Prime wk 0, A + B each Boost wks 2, 4 (ISCOMATRIX) 5 EI-Mutanttoxins 5 100 μg IM Prime wk 0, A + B each Boost wks 2, 4 (no adjuvant)Animals: 25 cynomolgus macaques The asterisk, “*”, in FIG. 16 refers tohaving only 4 cynos in the group for week 12, one cyno in the group wasterminally bled week at 8 Vaccination: IM, 0.5 mL per dose, at weeks 0,2, and 4. Mutant toxin compositions were prepared as described above.The mutant toxin compositions were formulated in ISCOMATRIX, exceptGroup 5 was formulated in buffer without adjuvant. Bleed: Weeks −2, 0,2, 3, 4, 6, 8, and 12. Euthanasia and terminal bleeds on animals withhighest C. difficile titers at week 8. Serum sample analysis: ProteinELISA and Neutralization assays

Results: FIG. 16 shows the anti-toxin neutralizing antibody responses inthese animals at weeks 0, 2, 3, 4, 6, 8, and 12. Anti-toxin A titerswere between 2-3 log₁₀ for all five groups after a single dose (week 2titers). These titers boosted after each subsequent dose for each group.In these animals, there was no drop in titer between weeks 3 and 4. Forall groups, the peak titers were between 4-5 log₁₀. At all time points,the group without ISCOMATRIX adjuvant (Group 5) had the lowest titers,indicating the utility of ISCOMATRIX at boosting the immune responses.The no-adjuvant control group (Group 5) reached peak titers at week 12,as did the group immunized with the high dose of EDC-inactivated mutanttoxins (Group 4); all other groups reached peak titers at week 6, twoweeks after the last dose. The titers in all groups boosted after thesecond dose (week 3 time point). As with the anti-toxin A responses, theanti-toxin B responses did not decrease from week 3 to week 4. After thethird dose (week 6 time point), the anti-toxin B neutralizing antibodytiters in all groups were between 3-4 log₁₀, except in the low doseformalin-inactivated group (Group 1) and the high dose EDC-inactivatedgroup (Group 4), both of which had titers just >4 log₁₀. The peak titerswere observed at week 12 for all groups except the low doseEDC-inactivated group (Group 3), which had peak titers at week 8. Allgroups had peak titers >4 log₁₀.

Example 31 Monoclonal Antibodies Production

Although toxins A and B share a lot of structural homology, theneutralizing activities of the antibodies were found to betoxin-specific. In this invention, several antibodies were identifiedthat are specific to individual toxin, and directed to various epitopesand functional domains, and have high affinity and potent neutralizingactivity toward native toxins. Antibodies were isolated from mice thatwere immunized with either a commercially available formalin inactivated(FI)-mutant toxin or recombinant holo-mutant toxin (SEQ ID NOs: 4 and 6)rendered non-toxic by introducing specific mutations in its catalyticsite for producing toxin A and B mAb, respectively. Epitope mapping ofthe antibodies showed that the vast majority of the mAb against toxin A(49 out of 52) were directed to the non-catalytic C terminal domain ofthe toxin.

Monoclonals against toxin B were targeted to three domains of theprotein. Out of a total of 17 toxin B specific mAb, 6 were specific toN-terminus (e.g., amino acids 1-543 of a wild-type C. difficile TcdB,such as 630), 6 to C-terminus (e.g., amino acids 1834-2366 of awild-type C. difficile TcdB, such as 630) and 5 to mid-translocationdomain (e.g., amino acids 799-1833 of a wild-type C. difficile TcdB,such as 630). The approach of using mutant C. difficile toxins (e.g.,SEQ ID NO: 4 and 6) as immunizing antigens thus offers a key advantageof presenting most, if not all, antigenic epitopes as compared to theformalin inactivation process that tend to adversely affect theantigenic structure of the mutant toxin.

Example 32 Characterization of Toxin a mAb, A3-25, which Includes aVariable Light Chain Having the Amino Acid Sequence of SEQ ID NO: 36 anda Variable Heavy Chain Having the Amino Acid Sequence of SEQ ID NO: 37

The mAb A3-25 was of particular interest since this antibody defied allattempts to define its immunoglobulin (Ig) isotyping using the commonlyavailable isotyping kits for IgG, IgM and IgA. Further analysis bywestern blot using Ig H-chain specific antisera showed that the A3-25 isof IgE isotype, a rare event in mAb production. This was furtherconfirmed by the nucleotide sequencing of mRNA isolated from A3-25hybridoma cells. The amino acid sequences deduced from the nucleotidesequences of the variable regions of H- and L-chain of A3-25 are shownin FIG. 17.

In order to further evaluate the A3-25 mAb in animal model for C.difficile infection and disease, its Ig isotype was changed to murineIgG1 by molecular grafting of the variable region of ε H chain onto themurine γ heavy chain according to the published methods.

Example 33 Neutralizing Ability and Epitope Mapping of Toxin SpecificAntibodies

Further, in an effort to identify functional/neutralizing antibodies,all monoclonals were evaluated for the ability to neutralize wild typetoxins in a standard cytopathic effect (CPE) assay or in a morestringent and quantitative assay based on measurement of ATP as cellviability indicator.

Out of a total of 52 toxin A specific antibodies, four mAb (A3-25,A65-33, A60-22 and A80-29 (Table 17 and FIG. 18) exhibited varied levelsof neutralizing activity. BiaCore competitive binding assay andhemagglutination inhibition (HI) assays were performed to map theantibody epitopes. Results indicated that these antibodies may betargeted to different epitopes of the toxin A protein (Table 17). Tofurther identify the location of binding sites on the protein, theantibodies were individually evaluated in western blot or dot blotassays using toxin fragments of known sequences. All 4 neutralizing mAbwere found to be directed to the C-terminus region of the toxin.

From a total of 17 toxin B specific antibodies, 9 were found to beneutralizing. Of the nine neutralizing mAb, six of them were directed tothe N-terminus and the other three to the translocation domain of the Btoxin (Table 18). Based on the Biacore competitive binding assay, thenine neutralizing monoclonal antibodies may be grouped into four epitopegroups as shown in FIG. 19.

TABLE 17 Characteristics of Selected Toxin A mAb Epitope Neutral-Hemagglu- Group izing tination Binding (Biacore) mAb # activityInhibition Specificity Ig Isotype 1 A3-25 + − C-teriminal IgE, κ 2A65-33 + − C-teriminal IgG2a, κ 3 A80-29 + + C-teriminal IgG1, κ NDA60-22 + + C-teriminal IgG1, κ 4 A64-6 − − In progress IgG1, κ A50-10 −− C-teriminal IgG1, κ A56-33 − − In progress IgG1, κ ND A1 − −N-terminal IgG1, κ

TABLE 18 Characteristics of Selected Toxin B mAb Epitope GroupNeutralizing Binding (Biacore) mAb # activity Specificity Ig isotype 1B2-31 + N-terminal IgG1, κ B5-40 IgG1, κ B8-26 IgG1, κ B70-2 IgG1, κ 2B6-30 + N-terminal IgG1, κ B9-30 IgG1, κ 3 B59-3 + Translocation IgG1, κB60-2 domain IgG1, κ 4 B56-6 + Translocation IgG1, κ B58-4 − domainIgG1, κ 5 B12-34 − C-terminal IgG1, κ B14-23 IgG1, κ B80-3 IgG1, κ 6B66-29 − C-terminal IgG1, κ 7 B84-3 − C-terminal IgG1, κ

Example 34 Identification of Novel Toxin A Antibodies Combinations withSignificantly Enhanced Neutralizing Activity

The four toxin A mAb (A3-25, A65-33, A60-22 and A80-29) showedincomplete or partial neutralization of toxin A when tested individuallyin the ATP based neutralization assay. The mAb A3-25 was the most potentantibody and the other three were less neutralizing with A80-29 barelyabove background (FIG. 18). However, when A3-25 was combined with eitherone of the other three mAbs, a synergistic effect in neutralization wasobserved in all three combinations which was far greater than the sumtotal of neutralization of individual antibodies as shown in FIG. 20A-C.In addition, all three combinations exhibited complete neutralizationcapability normally observed with anti-toxin A polyclonal antibodies.

Example 35 Identification of Novel Toxin B Antibodies CombinationsShowing Significantly Enhanced Neutralizing Activity

We also observed synergistic neutralization with the Toxin B mAbs fromthe different epitope groups identified by BiaCore analysis. Toxin B mAbB8-26, the most dominant mAb of group 1, was combined with multiple mAbsfrom group 3. The combinations were evaluated in a toxin B specificneutralization assay and the results are shown in FIG. 21 and Table 19.

TABLE 19 Neutralization of Toxin B with mAbs Neut titer mAb CPE ATPB8-26 alone 20,480 5,000 B59-3 alone 320 120 B60-2 alone 320 80 B8-26 +B59-3 655,360 ~60,000 B8-26 + B60-2 327,680 nd nd, not doneThe synergistic neutralizing effect was observed when B8-26 was combinedwith an epitope group 3 mAb (FIG. 21B), but not any other mAb (data notshown).

Example 36 In Vitro Screening by mAb for Safe and Efficacious MutantToxin Compositions

Genetic mutant toxins A and B of C. difficile (e.g., SEQ ID NO: 4 and 6)generated via genetic engineering showed residual cytotoxicity using anin vitro cytotoxicity assay. Although we have achieved a ˜4 logreduction in cytotoxicity for each mutant toxin C. difficile toxin(Table 20), further chemical inactivation of the mutant toxins, such aswith formalin treatment was preferred. However, chemical inactivationtreatments may be harsh and may adversely affect key antigenic epitopesof these toxins or mutant toxins.

TABLE 20 A Comparison of In Vitro Cytotoxicity of WT Toxin, TripleMutant Toxin, and Formalin-Inactivated (FI, from List Biological) WTtoxins (List Biological, commercial) Fold Reduction in TcdSource/treatment EC₅₀ ng/mL Cytotoxicity TcdA Toxin A WT 0.92 1 (SEQ IDNO: 1) Mutant toxin A Triple mutant 8600 9348 (SEQ ID NO: 4) Toxoid A(FI) Formalin treated, >20,000 >21,739 commercial TcdB Toxin B WT 0.0091 (SEQ ID NO: 2) Mutant toxin B Triple mutant 74 8222 (SEQ ID NO: 6)Toxoid B (FI) Formalin treated, 4300 477,778 commercial

For bioprocess optimization, a statistical design of experiment (DOE)was performed for the chemical inactivation of triple mutant Tcd A and B(1 mg/mL) using formalin and EDC/NHS treatment. To optimize formalininactivation of triple mutant TcdA, we varied concentrations offormalin/glycine (20-40 mM), pH (6.5-7.5), and temperature (25-40° C.).For triple mutant TcdB, we varied the formalin/glycine concentrationfrom 2 to 80 mM and the temperature and pH were 25° C. and 7.0respectively. The incubation time for all formalin treatments was 24hours. For the formalin inactivation, “40/40” in Tables 21 and 23represents the concentration of formalin and glycine used in thereaction. For EDC/NHS treatment, we varied the concentrations of EDC/NHSfrom 0.25 to 2.5 mg/mg of triple mutant TcdA and from 0.125 to 2.5 mg/mgof triple mutant TcdB and incubated for four hours at 25° C. At the endof the reactions, all samples were desalted in 10 mM phosphate, pH 7.0.After purification, the treated Tcds were analyzed for residualcytotoxicity and mAb recognition of epitopes by dot-blot analysis. Thegoal was to identify treatment conditions that reduce cytotoxicity tothe desired level (EC₅₀>1000 μg/mL) without negatively impactingepitopes recognized by a panel of neutralizing mAbs (++++ or +++). Thetreatment conditions (marked with a check mark “✓” in Tables 21-24)yielded potentially safe and efficacious immunogenic compositions thatretained reactivity to at least four neutralizing mAbs while exhibiting6-8 log₁₀ reduction in cytotoxicity, relative to the respectivewild-type toxin cytotoxicity. Select results are illustrated in Tables21 to 24. Additional data from varying treatment conditions on thetriple mutant toxins and the data from in vitro cytotoxicity and toxinneutralization assays are shown in Table 33 and Table 34. See also, forexample, Examples 20 and 21 above, which provide further detailsregarding preferred crosslinking treatment conditions of the mutanttoxins.

TABLE 21 Cytotoxicity and Neutralizing mAb Reactivity ofFormalin-inactivated Triple Mutant TcdA (SEQ ID NO: 4) Reactivity withmAb (dot blot, non-denaturing conditions) Chemical inactivationC-terminal (neut) reaction conditions on CPE N-terminal TranslocationA80- A3- A60- A65- Triple Mutant TcdA μg/mL Mab#6 Domain Mab# 102 29 2522 33 25° C., pH 6.5, 20/20 mM 250 ++++ ++++ ++++ ++++ ++++ ++++ 25° C.,pH 6.5, 40/40 mM ✓ >1000 ++++ ++++ ++++ ++++ ++++ ++++ 25° C., pH 7.5,40/40 mM ✓ >1000 ++++ ++++ ++++ ++++ ++++ ++++ 40° C., pH 6.5, 40/40mM >1000 ++ +++ ++++ ++++ ++++ ++++ 40° C., pH 7.5, 40/40 mM >1000 ++ ++++++ ++++ ++++ +++ None, Triple mutant toxin A 18.5-25 ++++ ++++ ++++++++ ++++ ++++ FI Toxoid A (List ND − − ++ ++ +++ + Biological)

TABLE 22 Cytotoxicity and Neutralizing mAb Reactivity of EDC-inactivatedTriple Mutant TcdA (SEQ ID NO: 4) Reactivity with mAb (dot blot,non-denaturing conditions) Chemical inactivation C-terminal (neut)reaction conditions on CPE N-terminal Translocation A80- A3- A60- A65-Triple Mutant TcdA μg/mL Mab#6 Domain Mab# 102 29 25 22 33 25° C., 0.25mg/mg, 4 hr ✓ >1000 ++++ ++++ ++++ ++++ ++++ ++++ 25° C., 0.5 mg/mg, 4hr ✓ >1000 ++++ ++++ ++++ ++++ ++++ ++++ 25° C., 1.25 mg/mg, 4 hr✓ >1000 +++ ++++ ++++ +++ ++++ ++++ 25° C., 2.5 mg/mg, 4 hr ✓ >1000 +++++++ ++++ +++ ++++ +++ None, Triple mutant TcdA 18.5-25 ++++ ++++ ++++++++ ++++ ++++ FI Toxoid A (List ND − − ++ ++ +++ + Biological)

TABLE 23 Cytotoxicity and Neutralizing mAb Reactivity of Formalin-inactivated Triple Mutant TcdB (SEQ ID NO: 6) mAb # mAb # Chemicalinactivation (N-terminal (mid-/C-terminal reaction conditions on CPE aa1-543) aa 544-2366) Triple Mutant TcdB (μg/mL) B8-26 B9-30 B56-6 B59-325° C., pH 7.0, >1000 ++++ ++++ ++++ +++ 80/80 mM, 24 hr ✓ 25° C., pH7.0, >1000 ++++ ++++ ++++ ++++ 40/40 mM, 24 hr ✓ 25° C., pH 7.0, 15.6++++ ++++ ++++ ++++ 10/10 mM, 24 hr 25° C., pH 7.0, <0.98 ++++ ++++ ++++++++ 2/2 mM, 24 hr None, Triple mutant 0.058 ++++ ++++ ++++ ++++ TcdB FIToxoid B (List ND +++ +++ +++ ++ Biological)

TABLE 24 Cytotoxicity and Neutralizing mAb Reactivity of EDC-inactivatedTriple Mutant TcdB (SEQ ID NO: 6) mAb # mAb # (N-terminal(mid-/C-terminal Chemical inactivation aa 1-543) aa 544-2366) reactionconditions on CPE 8-26 9-30 56-6 59-3 Triple Mutant TcdB (μg/mL) B8-26B9-30 B56-6 B59-3 25° C., 0.125 3.9 ++++ ++++ ++++ ++++ mg/mg, 4 hr 25°C., 0.25 250 ++++ ++++ ++++ ++++ mg/mg, 4 hr 25° C., 0.5 >1000 ++++ ++++++++ ++++ mg/mL, 4 hr ✓ 25° C., 1.25 >1000 ++++ +++ +++ +++ mg/mg, 4 hr✓ 25° C., 2.5 >1000 ++++ +++ +++ +++ mg/mg, 4 hr ✓ None, Triple mutant0.058 ++++ ++++ ++++ ++++ TcdB FI Toxoid B (List ND +++ +++ +++ ++Biological)

TABLE 33 Reactivity with mAb (dot blot, non-denaturing Cyto Assayconditions) Mutant toxin A (EC50) N- Translocation (SEQ ID NO: CPE; CPE,terminal Domain Mab# C-terminal (neut) Sample # 4) Sample ID 24 h μg/mL72 h μg/mL Mab#6 102 80-29 3-25 60-22 65-33 1 L44166-157A >1000 >1000++++ ++++ ++++ +++ ++++ ++++ 2 L44166-157B >1000 >1000 +++ ++++ ++++ +++++++ ++++ 3 L44166-157C >1000 >1000 +++ +++ ++++ +++ ++++ ++++ 4L44166-157D >1000 >1000 +++ +++ ++++ +++ ++++ ++++ 5L44905-160A >1000 >1000 ++ ++ ++++ ++ ++++ ++++ 6 L44166-166 >1000 >1000++++ ++++ ++++ ++++ ++++ ++++ 7 L44905-170A ND >1000 + + ++ ++ ++ + 8L44897-61 >1000 ND +++ ++ ++++ ++++ ++++ ++++ 9 L44897-63 >1000 ND +++++++ ++++ +++ ++++ ++++ 10 L44897-72 250 ND ++++ ++++ ++++ ++++ ++++ ++++Tube#1 11 L44897-72 >1000 ND ++++ ++++ ++++ ++++ ++++ ++++ Tube#2 12L44897-72 >1000 ND +++ +++ ++++ ++++ ++++ ++++ Tube#3 13 L44897-72 >1000ND +++ ++++ ++++ ++++ ++++ ++++ Tube#4 14 L44897-72 >1000 ND +++ ++++++++ ++++ ++++ ++++ Tube#5 15 L44897-75 >1000 ND +++ ++++ ++++ ++++ ++++++++ Tube#6 16 L44897-75 >1000 ND ++++ ++++ ++++ ++++ ++++ ++++ Tube#717 L44897-75 >1000 ND ++++ ++++ ++++ ++++ ++++ ++++ Tube#8 18L44897-75 >1000 ND ++ +++ ++++ ++++ ++++ ++++ Tube#9 19 L44897-75 >1000ND ++++ ++++ ++++ ++++ ++++ ++++ Tube#10 20 L44897-75 >1000 ND ++ ++++++ ++++ ++++ +++ Tube#11 21 L44897-101 23.4 <7.8 ++++ ++++ ++++ ++++++++ ++++ (pre- modification) TxA control 22 L44897-101, 2 hr 187.5155.9 +++ ++++ ++++ ++++ ++++ ++++ 23 L44897-101, 4 hr 375 380.3 +++++++ ++++ ++++ ++++ ++++ 24 L44897-101, 6 hr 500 429.6 +++ ++++ ++++++++ ++++ ++++ 25 L44897 102, >1000 >1000 ++ ++++ ++++ ++++ ++++ ++++ 24hr 26 L44897-103, >1000 >1000 + +++ +++ ++++ ++++ +++ 51 hr 27L44897-104, >1000 >1000 − +++ +++ +++ +++ +++ 74 hr 28L44897-105, >1000 >1000 − ++ ++ +++ +++ ++ 120 hr 29L44980-004 >1000 >1000 ++++ ++++ ++++ ++++ ++++ ++++ 30 Reaction #1 750ug/mL ND ND ++ ++ +++ +++ ++ Week 0, 25C 31 Reaction #1 375 ug/mL ND ND+++ +++ +++ +++ +++ Week 1, 25C 32 Reaction #1 375 ug/mL ND ND +++ ++++++ +++ +++ Week 2, 25C 33 Reaction #1 375 ug/mL ND ND +++ +++ +++ ++++++ Week 3, 25C 34 Reaction #1 250 ug/mL ND ND +++ +++ +++ +++ +++ Week4, 25C 35 Reaction #1 93.8 ug/mL  ND ND ++++ ++++ ++++ ++++ ++++ Week 3,37C 36 Reaction #2 375 ug/mL ND ND +++ +++ +++ +++ +++ Week 0, 25C 37Reaction #2 375 ug/mL ND ND ++++ ++++ ++++ ++++ ++++ Week 1, 25C 38Reaction #2 750 ug/mL ND ND ++ ++ ++ +++ ++ Week 2, 25C 39 Reaction #2250 ug/mL ND ND +++ +++ +++ ++++ +++ Week 3, 25C 40 Reaction #2 250ug/mL ND ND +++ +++ +++ ++++ +++ Week 4, 25C 41 Reaction #2 187.5ug/mL   ND ND +++ +++ ++++ ++++ +++ Week 3, 37C 42 TxA Control 18.8ug/mL  ND ND ++++ ++++ ++++ ++++ ++++ Week 3, 25C 43 TxA Control 25ug/mL ND ND ++++ ++++ ++++ ++++ ++++ Week 3, 37C 44 L44897-116-6 >2000ug/mL  ND ND ++ ++ ++ +++ ++ 29.5 hrs 45 L44897-116-7 >2000 ug/mL  ND ND++ ++ ++ +++ ++ 57.5 hrs 46 L44897-116-8 >2000 ug/mL  ND ND + + + +++ +79.5 hrs 47 L44897-116-9 >2000 ug/mL  ND ND ++ ++ ++ +++ ++ 123.5 hrs 48L44897-139 >1000 ND ++ ++++ ++++ ++++ ++++ ++++ 49 L44166-204 >1000 ND++++ ++++ ++++ ++++ ++++ ++++

Chemical Crosslinking Reaction Conditions for the Samples of TripleMutant Toxin a (SEQ ID NO: 4) Referenced in Table 33

Samples 1-4 were modified with EDC/NHS. Conditions: 30° C., 20 mMMES/150 mM NaCl pH 6.5. Reactions were initiated by addition of EDC.After 2 hours reaction, samples A, B, and C had 1 M glycine added to 50mM glycine final concentration. Sample D had no glycine added. Thereactions were set up with different weight ratios of Mutant toxin A(SEQ ID NO: 4):EDC:NHS as indicated below.

-   -   1 L44166-157A 1:0.25:0.25 w:w:w    -   2 L44166-157B 1:1.25:1.25    -   3 L44166-157C 1:2.5:2.5    -   4 L44166-157D 1:2.5:2.5

Sample 5 L44905-160A 80 mM HCHO, 80 mM glycine, 80 mM NaPO4 pH 7, 1mg/mL Mutant toxin A (SEQ ID NO: 4) Protein, 48 hrs reaction at 25° C.

Sample 6 L44166-166 EDC/NHS modification of Mutant toxin A (SEQ ID NO:4) at 25° C. in 20 mM MES/150 mM NaCl pH 6.5. Mutant toxin A (SEQ ID NO:4):EDC:NHS=1:0.5:0.5. Reaction initiated by addition of EDC. After 2hours reaction, 1M glycine added to 0.1 M glycine final concentrationand further 2 hour incubation. After this time, reaction bufferexchanged into 1×PBS on Sephadex G25.

Sample 7 L44905-170A 80 mM HCHO, 80 mM glycine, 80 mM NaPO₄ pH 7, 1mg/mL Mutant toxin A (SEQ ID NO: 4) Protein, 48 hrs reaction at 35 C.This formalin reaction was directed at producing excessive crosslinkingso that antigen binding would be severely diminished.

Sample 8 L44897-61 32 mM HCHO/80 mM glycine, 72 hrs reaction at 25° C.

Sample 9 L44897-63 80 mM HCHO/80 mM glycine, 72 hrs reaction at 25° C.The following reactions all had 24 hrs reaction time.

Sample 10 L44897-72 Tube#1 25° C., 80 mM NaPi pH 6.5, 20 mM HCHO/20 mMglycine

Sample 11 L44897-72 Tube#2 25° C., 80 mM NaPi pH 6.5, 40 mM HCHO/40 mMglycine

Sample 12 L44897-72 Tube#3 32.5° C., 80 mM NaPi pH 7.0, 30 mM HCHO/30 mMglycine

Sample 13 L44897-72 Tube#4 32.5° C., 80 mM NaPi pH 7.0, 30 mM HCHO/30 mMglycine Sample 14 L44897-72 Tube#5 32.5° C., 80 mM NaPi pH 7.0, 30 mMHCHO/30 mM glycine

Sample 15 L44897-75 Tube#6 25° C., 80 mM NaPi pH 7.5, 20 mM HCHO/20 mMglycine

Sample 16 L44897-75 Tube#7 25° C., 80 mM NaPi pH 7.5, 40 mM HCHO/40 mMglycine

Sample 17 L44897-75 Tube#8 40° C., 80 mM NaPi pH 6.5, 20 mM HCHO/20 mMglycine

Sample 18 L44897-75 Tube#9 40° C., 80 mM NaPi pH 6.5, 40 mM HCHO/40 mMglycine

Sample 19 L44897-75 Tube#10 40° C., 80 mM NaPi pH 7.5, 20 mM HCHO/20 mMglycine

Sample 20 L44897-75 Tube#11 40° C., 80 mM NaPi pH 7.5, 40 mM HCHO/40 mMglycine

The following 8 samples were reacted at 25° C. for the indicated timesin 80 mM NaPi pH 7.0 containing 78 mM HCHO and 76 mM glycine

Sample 21 L44897-101 (pre-modification) TxA control time zero controlsample, not modified or exposed to HCHO/glycine

Sample 22 L44897-101, 2 hr

Sample 23 L44897-101, 4 hr

Sample 24 L44897-101, 6 hr

Sample 25 L44897 102, 24 hr

Sample 26 L44897-103, 51 hr

Sample 27 L44897-104, 74 hr

Sample 28 L44897-105, 120 hr

Sample 29 (L44980-004) was EDC/NHS modified Mutant toxin A (SEQ ID NO:4) (triple mutant toxin A (SEQ ID NO: 4)-EDC). Reaction conditions are:25° C., buffer was 20 mM MES/150 mM NaCl pH 6.6. Triple mutant toxin A(SEQ ID NO: 4):EDC:NHS=1:0.5:0.5 w:w:w. Reaction initiated by additionof EDC. After 2 hours reaction, glycine added to 0.1 M finalconcentration and reacted further 2 hours at 25 C. Reaction terminatedby desalting on Sephadex G25.

The following 12 samples and 2 controls were reversion experiments wheresamples were incubated at 25° C. and 37° C.

Reaction 1=25° C., 80 mM NaPi pH 7.0, 40 mM HCHO only (no glycine), 24hour reaction.Reaction 2=25° C., 80 mM NaPi pH 7.0, 40 mM HCHO/40 mM glycine, 24 hourreaction

Sample Reaction 30 Reaction #1 Week 0, 25° C. 31 Reaction #1 Week 1, 25°C. 32 Reaction #1 Week 2, 25° C. 33 Reaction #1 Week 3, 25° C. 34Reaction #1 Week 4, 25° C. 35 Reaction #1 Week 3, 37° C. 36 Reaction #2Week 0, 25° C. 37 Reaction #2 Week 1, 25° C. 38 Reaction #2 Week 2, 25°C. 39 Reaction #2 Week 3, 25° C. 40 Reaction #2 Week 4, 25° C. 41Reaction #2 Week 3, 37° C. 42 TxA Control Week 3, 25° C. 43 TxA ControlWeek 3, 37° C.The next 4 samples were generated by reaction for the indicated times at25° C. in 80 mM NaPi pH 7.0, 40 mM HCHO/40 mM glycine

-   -   44 L44897-116-6 29.5 hrs    -   45 L44897-116-7 57.5 hrs    -   46 L44897-116-8 79.5 hrs    -   47 L44897-116-9 123.5 hrs

Sample 48 L44897-139 48 hrs reaction at 25° C., 80 mM NaPi pH 7.0, 40 mMHCHO/40 mM glycine.

Sample 49 L44166-204 EDC/NHS modification of Mutant toxin A (SEQ ID NO:4). 25 C, buffer 1×PBS pH7.0. Mutant toxin A (SEQ ID NO:4):EDC:NHS=1:0.5:0.5 w:w:w. 2 hours reaction with EDC/NHS, then 1 Mglycine added to 0.1 M final concentration and further 2 hours reaction.Buffer exchanged on Sephadex G25 into 20 mM L-histidine/100 mM NaCl pH6.5.

TABLE 34 Reactivity with neut mAb (dot blot, non-denaturing conditions)mAb # (N- mAb # (mid-/C- Strong terminal aa 1-543) terminal aa 544-2366)reactivities Mutant toxin B Cyto Assay (EC50) 8-26 56-6 to Sample IDCPE; 24 h ATP, 72 h 9-30 59-3 all 4 mAbs L44905-86-01 <0.1 μg/mL <0.1μg/mL ++++ ++++ ++++ ++++ ✓ Triple mutant toxin B (SEQ ID NO: 6),Untreated Control L44905-86-02 ≧100 μg/mL 2.2 μg/mL ++++ ++++ ++++ ++++✓ Triple mutant toxin B (SEQ ID NO: 6), Rxn1, 10° C., day1L44905-86-03 >100 μg/mL >100 μg/mL +++ ++++ ++ +++ ✓* Triple mutanttoxin B (SEQ ID NO: 6), Rxn1, 25° C., day1 L44905-86-04 >100 μg/mL 5.2μg/mL ++++ ++++ ++++ ++++ ✓ Triple mutant toxin B (SEQ ID NO: 6), Rxn2,10° C., day1 L44905-86-05 >100 μg/mL >100 μg/mL ++++ ++++ ++ +++ ✓*Triple mutant toxin B (SEQ ID NO: 6), Rxn2, 25° C., day1L44905-86-06 >100 μg/mL >100 μg/mL ++++ − ++++ +++ Triple mutant toxin B(SEQ ID NO: 6), Rxn3, 10° C., day1 L44905-86-07 >100 μg/mL >100 μg/mL++++ − ++++ +++ Triple mutant toxin B (SEQ ID NO: 6), Rxn3, 25° C., day1L44905-86-08 >100 μg/mL >100 μg/mL ++++ ++++ ++++ +++ ✓ Triple mutanttoxin B (SEQ ID NO: 6), Rxn1, 10° C., day5 L44905-86-09 >100 μg/mL >100μg/mL ++ ++ − + Triple mutant toxin B (SEQ ID NO: 6), Rxn1, 25° C., day5L44905-86-10 >100 μg/mL >100 μg/mL ++++ ++++ ++++ ++++ ✓ Triple mutanttoxin B (SEQ ID NO: 6), Rxn2, 10° C., day5 L44905-86-11 >100 μg/mL >100μg/mL ++ ++++ − + Triple mutant toxin B (SEQ ID NO: 6), Rxn2, 25° C.,day5 L44905-86-12 >100 μg/mL >100 μg/mL ++++ − ++++ +++ Triple mutanttoxin B (SEQ ID NO: 6), Rxn3, 10° C., day5 L44905-86-13 >100 μg/mL >100μg/mL ++++ − ++++ ++++ Triple mutant toxin B (SEQ ID NO: 6), Rxn3, 25°C., day5 L44905-86-14 >100 μg/mL >100 μg/mL ++++ ++++ ++++ ++++ ✓ Triplemutant toxin B (SEQ ID NO: 6), Rxn1, 10° C., day7 L44905-86-15 >100μg/mL >100 μg/mL +++ ++++ − + Triple mutant toxin B (SEQ ID NO: 6),Rxn1, 25° C., day7 L44905-86-16 >100 μg/mL >100 μg/mL +++ ++++ +++ ++++✓ Triple mutant toxin B (SEQ ID NO: 6), Rxn2, 10° C., day7L44905-86-17 >100 μg/mL >100 μg/mL ++ ++ − + Triple mutant toxin B (SEQID NO: 6), Rxn2, 25° C., day7 L44905-86-18 >100 μg/mL >100 μg/mL ++++ −++++ ++++ Triple mutant toxin B (SEQ ID NO: 6), Rxn3, 10° C., day7L44905-86-19 >100 μg/mL >100 μg/mL +++ − ++ ++ Triple mutant toxin B(SEQ ID NO: 6), Rxn3, 25° C., day7 L34346-30A >100 μg/mL >100 μg/mL ++++++++ ++++ ++++ ✓ L34346-30B >100 μg/mL >100 μg/mL +++ ++++ ++++ ++++ ✓Commercial, FI ND ND ++++ ++++ ++++ ++++ ✓ Toxoid B (List Biologicals)Commercial, 22.5 pg/mL 7.8 pg/mL +++ ++ +++ +++ ✓ Control Toxin B wt(List Biologicals) Control, 78 ng/mL 72 ng/ml +++ ++ ++++ +++ ✓recombinant triple mutant toxin B (SEQ ID NO: 6)

Chemical Crosslinking Reaction Conditions for the Samples of MutantToxin B Referenced in Table 34

Triple mutant toxin B (SEQ ID NO: 6) was chemically crosslinked andtested according to the following reaction conditions. The L44905-86samples were tested in an experiment involving three formalin reactionvariations and two incubation temperatures. Each day, 6 samples weretaken for a total of 18 samples. The first sample in the list is theuntreated control (which makes 19 samples total). The untreated controlincluded an untreated triple mutant toxin B polypeptide (SEQ ID NO: 6).

Reaction1 (“R×n1”)=80 mM HCHO, 80 mM glycine, 80 mM NaPO4 pH 7, 1 mg/mLTriple mutant toxin B (SEQ ID NO: 6) Protein

Reaction2 (“R×n2”)=80 mM HCHO, No glycine, 80 mM NaPO4 pH 7, 1 mg/mLTriple mutant toxin B (SEQ ID NO: 6) Protein

Reaction3 (“R×n3”)=80 mM HCHO, No glycine, 80 mM NaPO4 pH 7, 1 mg/mLTriple mutant toxin B (SEQ ID NO: 6) Protein+Cyanoborohydride capping.Cyanoborohydride Capping involved 80 mM CNBrH₄ added to desalted finalreaction and incubated 24 hr at 36° C.

For Sample L34346-30A 0.5 g EDC and NHS per gram of triple mutant toxinB (SEQ ID NO: 6), 4 hours at 30° C., in 20 mM MES, 150 mM NaCl, pH 6.5.

For Sample L34346-30B 0.5 g EDC and NHS per gram of triple mutant toxinB (SEQ ID NO: 6), 2 hours at 30° C. followed by addition of glycine(final concentration of g/L) and incubated another 2 hours at 30° C., in20 mM MES, 150 mM NaCl, pH 6.5. The only difference between the tworeactions for L34346-30A and L34346-30B is the addition of glycine toreaction L34346-30B.

Example 37 Antibodies Induced by Immunogenic Compositions are Capable ofNeutralizing Toxins from Various C. difficile Strains

To assess whether antibodies induced by the immunogenic compositionsincluding the mutant toxin drug substances can neutralize a broadspectrum of diverse toxin sequences, strains representing diverseribotypes and toxinotypes were sequenced to identify the extent ofgenetic diversity among the various strains compared to the mutant toxindrug substances. Culture supernatants containing secreted toxins fromthe various strains were then tested in an in vitro neutralization assayusing sera from immunized hamsters to determine the coverage of theimmunogenic composition and to determine the ability of the immunogeniccomposition to protect against diverse toxins from circulating clinicalstrains.

Both HT-29 cells (colon carcinoma cell line) and IMR-90 cells were usedto test the neutralization of toxins expressed from CDC strains. HT-29cells are more sensitive to TcdA; the EC₅₀ of the purified TcdA in thesecells is 100 μg/mL as compared to 3.3 ng/mL for TcdB. On the other handIMR-90 cells are more sensitive to TcdB; the EC₅₀ of the purified TcdBin these cells ranges between 9-30 μg/mL as compared to 0.92-1.5 ng/mLfor TcdA. The assay specificity for both TcdA and TcdB in these celllines was confirmed by using both polyclonal and monoclonaltoxin-specific antibodies. For assay normalization, culture filtrates ofthe 24 CDC isolates were tested at a concentration four times theirrespective EC₅₀ value. Three of the strains had toxin levels that weretoo low for testing in the neutralization assay.

Twenty-four strains representing diverse ribotypes/toxinotypes coveringgreater than 95% of the circulating strains of C. difficile in the USAand Canada were obtained from the CDC. Among these isolates were strainsrepresenting ribotypes 027, 001 and 078, three epidemic strains of CDADin the United States, Canada and UK. Strains 2004013 and 2004118represented ribotype 027; strain 2004111 represented ribotype 001 andstrains 2005088, 2005325 and 2007816 represented ribotype 078. Toidentify the extent of genetic diversity between the disease-causingclinical isolates and the 630 strain, the toxin genes (tcdA and tcdB)from these clinical strains were fully sequenced. See Table 35. Theamino acid sequences of the toxins were aligned using ClustalW in theMegalign™ program (DNASTAR® Lasergene®) and analyzed for sequenceidentity. For tcdA, genomic alignment analysis showed that all of theclinical isolates and strain 630 shared overall about 98-100% amino acidsequence identity. The C-terminal portion of the tcdA gene was slightlymore divergent. The same analysis was performed for the tcdB gene whichexhibited greater sequence divergence. Notably strains 2007838/NAP7/126and 2007858/NAP1/unk5 displayed the most divergent patterns from the 630strain in the N terminal (79-100%) and the C terminal domains (88-100%;data not shown).

A hamster serum pool (HS) was collected from the Syrian golden hamstersthat were immunized with an immunogen including mutant TcdA (SEQ ID NO:4) and mutant TcdB (SEQ ID NO: 6), wherein the mutant toxins wereinactivated with EDC, according to, for example, Example 29, Table 15,described above, and formulated with aluminum phosphate. The results inTable 35 show that at least toxin B from the respective culturesupernatants were neutralized, in an in vitro neutralization assay, bysera from the immunized hamsters.

TABLE 35 Description of C. difficile strains from CDC and Ability ofImmune Hamster Sera to Neutralize Various Toxins Neutralized by HamsterStrain PFGE Type Ribotype Sera 2005088 NAP7 78 yes 2007816 NAP7- 78 yesrelated 2005325 NAP7 78 yes 2004013 NAP1 27 yes 2007886 NAP1 yes 2008222NAP4 77 yes 2004206 NAP4 154  yes 2005283 NAP5 Unk3 Not tested^(b)2009141 NAP2 yes 2007838 NAP7 126  yes 2004111 NAP2  1 yes 2007070 NAP1070 yes 2006017 NAP12 15 yes 2009078 NAP11 106  Not tested^(b) 2007217NAP8 126  yes 2006376 NAP9 17 yes 2007302 NAP11 Unk2 yes 2004118 NAP1 27yes 2005022 NAP3 53 yes 2009292 NAP1 yes 2004205 NAP6  2 yes 2007858NAP1 Unk5 yes 2009087 NAP11 106  Not tested^(b) 2005359 NAP1- yesrelated ^(b)Toxin levels were too low to perform the neutralizationassay.

FIG. 23 depicts the results of the neutralization assay using toxinpreparations from various C. difficile strains on IMR-90 cells. The datashow TcdB neutralizing antibodies in the hamster antisera were capableof neutralizing toxins from all 21 isolates tested, includinghypervirulent strains and a TcdA-negative, TcdB-positive strain. Atleast 16 different strains of C. difficile were obtained from the CDC(Atlanta, Ga.)(previously described) and were cultured in C. difficileculture media under suitable conditions as known in the art and asdescribed above. Culture supernatants containing the secreted toxinswere analyzed to determine their cytotoxicity (EC₅₀) on IMR-90monolayers and subsequently tested in a standard in vitro neutralizationassay at 4 times the EC₅₀ using various dilutions of sera from hamstersimmunized with mutant toxin A drug substance and mutant toxin B drugsubstance, formulated with aluminium phosphate. Crude toxin obtainedfrom culture supernatants of each strain and purified toxin (commercialtoxin obtained from List Biologicals)(not purified from respectivesupernatants) were tested for cytotoxicity to IMR-90 cells using the invitro cytotoxicity assay described above.

In FIGS. 23A-K, the graphs show results from in vitro cytotoxicity tests(previously described) in which the ATP levels (RLUs) are plottedagainst increasing concentrations of: C. difficile culture media and thehamster serum pool (▪); crude toxin and the hamster serum pool ();purified toxin and the hamster serum pool (▴); crude toxin (▾), control;and purified toxin (♦), control. The toxins from the respective strainswere added to the cells at 4×EC₅₀ values.

As shown in FIGS. 23A-K, the hamsters that received the describedimmunogen surprisingly developed neutralizing antibodies that exhibitedneutralizing activity against toxins from at least the following 16different CDC strains of C. difficile, in comparison to the respectivetoxin only control: 2007886 (FIG. 23A); 2006017 (FIG. 23B); 2007070(FIG. 23C); 2007302 (FIG. 23D); 2007838 (FIG. 23E); 2007886 (FIG. 23F);2009292 (FIG. 23G); 2004013 (FIG. 23H); 2009141 (FIG. 23I); 2005022(FIG. 23J); 2006376 (FIG. 23K). See also Table 35 for additional C.difficile strains from which toxins were tested and were neutralized bythe immunogenic composition including a mutant toxin A drug substanceand mutant toxin B drug substance, formulated in aluminum phosphate.

In another study, culture supernatants containing secreted toxins fromthe various C. difficile strains (obtained from the CDC and from LeedsHospital, UK) were tested in the in vitro neutralization assay usingsera from hamsters that were administered with mutant toxin A drugsubstance and mutant toxin B drug substance, formulated with Alhydrogel.See Table 36 for the experimental design. The results are shown in Table37 and Table 38.

TABLE 36 Experimental design Assay In assay using HT-29 cells: Rabbitanti-serum (Anti-Toxin A Control polyclonal Fitzgerald Industries,#70-CR65) and Reference Toxin A (wild-type toxin A from ListBiologicals) In assay using IMR-90 cells: Rabbit anti-serum (Anti-ToxinB polyclonal Meridian Life Science, #B01246R) and Reference Toxin B(wild-type toxin B from List Biologicals) Sample In assay using HT-29cells: HS serum + Reference Toxin A Controls In assay using IMR-90cells: HS serum + Reference Toxin B HS serum + 630 wt toxin HS serum +Culture media of IMR-90 or HT-29 cell line HS serum + culturesupernatant of VPI11186 Test HS + respective C. difficile culturesupernatant Sample Source of Animals administered with mutant toxin Adrug substance and Hamster mutant toxin B drug substance formulated withAlhydrogel antiserum (HS)

TABLE 37 Immunogenic Composition-induced Antibodies Neutralized Toxin Aand Toxin B from Various Wild-type C. difficile Strains from the CDC,including Hypervirulent strains Neutralized by Neutralized by Cdiff PFGEOther Typing HS (IMR-90, HS (HT-29, Strain Type Ribotype ToxinotypeMethod Toxin B) Toxin A) 2004111 NAP2 1 0 Respective toxin Yes Yes2009141 NAP2 0 sequence has 100% Yes Yes 2006017 NAP12 15 0 Homology totoxin Yes Yes 2007302 NAP11 Unk2 0 from Strain 630 Yes Yes 2009087 NAP11106 0 Yes Yes 2005022 NAP3 53 0 Yes Yes 2005283 NAP5 Unk3 0 Yes Yes2009078 NAP5 53 0 Yes Yes 2004206 NAP4 154 0 Yes Yes 2008222 NAP4 77 0Yes Yes 2004205 NAP6 2 0 Yes Yes 2007070 NAP10 70 0 Yes Yes 2006376 NAP917 VIII txnA−/txnB+ Yes N/A 2007816 NAP7- 78 V Increasing Yes Yesrelated prevalence in US 2007838 NAP7 126 and Europe Yes Yes 2005088NAP7 78 Yes Yes 2005325 NAP7 78 Yes Yes 2007217 NAP8 126 Yes Yes 2004013NAP1 27 III Hypervirulent Yes Yes 2004118 NAP1 27 NAP1/027/III Yes Yes2009292 NAP1 Yes Yes 2005359 NAP1- Yes Yes related 2007858 NAP1 Unk5IX/XXIII Other Yes Yes 2007886 NAP1 IX/XXIII Yes Yes

TABLE 38 Immunogenic Composition-induced Antibodies Neutralized Toxin Aand Toxin B from Various Wild-type C. difficile Strains from Europe,including Hypervirulent strains Neutralized Neutralized by HS by HS Cdiff PFGE Other Typing Toxin (IMR-90, (HT-29, Strain Type Method typeToxin B) Toxin A) 001 NAP2 Toxinotype 0 0 Yes Yes 002 NAP6 Strains YesYes 012 NAPCR1 Yes Yes (004) 014 UK Yes Yes 015 NAP12 Yes Yes 020 NAP4Yes Yes 029 UK Yes Yes 046 UK Yes Yes 053 NAP5 Yes Yes 059 UK Yes Yes077 UK Yes Yes 078 UK Yes Yes 081 UK Yes Yes 087 UK Yes Yes 095 UK YesYes 106 UK Yes Yes 117 UK Yes Yes 017 NAP9 txnA−/txnB+ VIII Yes NA 027NAP1 Hypervirulent III Yes Yes 075 UK Yes Yes 003 NAP10 Other I Yes Yes023 UK IV Yes Yes 070 UK XIII Yes Yes 126 UK UK Yes Yes 131 UK UK InProgress Yes Wild-type C. difficile strains obtained from LeedsHospital, UK. “UK” = unknown status NA, not applicable; strain does notmake toxin A; was not tested in Toxin A neutralization assay

Example 38 Peptide Mapping of EDC/NHS Triple Mutant Toxins

To characterize the EDC/NHS inactivated triple mutant toxins, peptidemapping experiments were performed on four lots of EDC/NHS-treatedtriple mutant toxin A (SEQ ID NO: 4) and four lots of EDC/NHS-treatedtriple mutant B (SEQ ID NO: 6). After digesting the mutant toxins withtrypsin, the resulting peptide fragments were separated usingreverse-phase HPLC. Mass spectral analysis was used to identifymodifications that occur as a result of the inactivation process. Forboth mutant toxin A drug substance and mutant toxin B drug substance,greater than 95% of the theoretical tryptic peptides were identified.Crosslinks and glycine adducts (glycine was used as the capping agent)were identified. In both mutant toxin A drug substance and mutant toxinB drug substance, beta-alanine adducts were also observed. Without beingbound by mechanism or theory, the beta-alanine adducts appear to resultfrom the reaction of three moles of NHS with one mole of EDC which formsNHS activated beta-alanine. This molecule can then react with lysinegroups to form beta-alanine adducts (+70 Da). In the EDC/NHS-treatedtriple mutant toxin B samples, low levels (0.07 moles/mole protein) ofdehydroalanine (−34 Da) were also observed. Dehydroalanine is a resultof de-sulfonation of a cysteine residue. The same type and degree ofmodification was observed in all four batches of each mutant toxin,indicating that the process produces a consistent product. Peptidemapping (at greater than 95% sequence coverage) confirms thatmodifications are present. A summary of the modifications are shown inTable 39. See also FIGS. 24-25. In addition, the size and chargeheterogeneity of the triple mutant toxin A drug substance and of thetriple mutant toxin B drug substance increased, as compared to the sizeand charge heterogeneity of the respective triple mutant toxin A andtriple mutant toxin B in the absence of chemical inactivation. As aresult, the size-exclusion chromatography (SEC) and anion-exchangechromatography (AEX) profiles had relatively broad peaks (data notshown).

TABLE 39 Summary of Modifications Observed in Mutant Toxin DrugSubstances Moles # of modified/ Modified Total # of Degree of moleModification Residues Residues Modification protein Mutant toxin A drugsubstance Crosslink 2 313 Asp/Glu 16-40% 0.6 Glycine moiety 8 313Asp/Glu 10-53% 2.2 Beta Alanine 19 233 Lys 10-60% 4.7 moiety Mutanttoxin B drug substance Crosslink 3 390 Asp/Glu 11-63% 0.8 Glycine moiety23 390 Asp/Glu 10-31% 3.9 Beta Alanine 10 156 Lys 12-42% 2.6 moietydehydroalanine 2 8 Cys 1.0-3.5%  .07The degree of modification is calculated by dividing the HPLC area ofmodified peptide by the HPLC area of the native+modified peptide.

Example 39 Drug Product Production

The C. difficile immunogenic composition (drug product) contains twoactive pharmaceutical ingredients (mutant toxin A drug substance andmutant toxin B drug substance). An exemplary drug product is alyophilized formulation containing 10 mM Tris buffer pH 7.4, 4.5% (w/w)trehalose dihydrate, and 0.01% (w/v) polysorbate 80, including each of amutant toxin A drug substance and a mutant toxin B drug substance. SeeTable 40. The immunogenic composition is prepared for injection byresuspending the lyophilized vaccine either with diluent or with diluentcontaining Alhydrogel. The placebo will include a sterile normal salinesolution for injection (0.9% sodium chloride).

TABLE 40 Component Selected Formulation dosage form Lyophilized Antigendose per 0.5 mL 25, 50, 100 μg of each EDC/NHS- treated triple mutanttoxin A (SEQ ID NO: 4) and EDC/NHS-treated triple mutant toxin B (SEQ IDNO: 6) pH 7.4 ± 0.5 Buffer 10 mM Tris Stabilizer/Bulking agent 4.5%Trehalose dihydrate (3-6%) Surfactant 0.01% Polysorbate 80(0.005-0.015%) Container closures 2 mL 13 mm Type 1 flint glass Vial,Blowback, West - Flurotec

Buffer Preparation

Water for injection (WFI) is added to a compounding vessel. Whilemixing, the excipients are added and dissolved until into solution. ThepH is measured. If required, the pH is adjusted to 7.4±0.1 with HCl. Thesolution is diluted to the final weight with WFI then filtered using a0.22 μm Millipore Express SHC XL150 filter. A pre-filtration bioburdenreduction sample is taken prior to filtration. The filtered buffer issampled for osmolality and pH.

Formulation Preparation

The thawed mutant toxin Drug Substances are pooled in the formulationvessel based on the precalculated amounts in the following order ofoperation: 50% of the target dilution buffer volume to achieve 0.6 mg/mLis added to the vessel first, followed by addition of mutant toxin Adrug substance and mixed for 5 minutes at 100 rpm. Mutant toxin B drugsubstance is then added to the vessel and the solution is furtherdiluted to 0.6 mg/mL dilution point and then mixed for another 5 minutesat 100 rpm. A sample is removed and tested for total mutant toxinconcentration. The solution is diluted to 100 percent volume based onthe in-process mutant toxin concentration value then mixed for 15minutes at 100 rpm. The formulated drug product is sampled for pH andbioburden pre-filtration. The formulated drug product is then filteredusing a Millipore Express SHC XL150 for overnight storage, or brought tothe filling line for sterile filtration.

The formulated bulk is brought to the filling area, sampled forbioburden, and then sterile filtered with two in-series MilliporeExpress SHC XL150 filters. The formulated bulk is filled intodepyrogenated glass vials at a target fill volume of 0.73 mL. The filledvials are partially stoppered and then loaded into the freeze dryer. Thelyophilization cycle is executed as shown in Table 41. At the completionof cycle, the lyophilization chamber is back-filled with nitrogen to 0.8atm and then the stoppers are fully seated. The chamber is unloaded andthe vials are capped using flip-off seals.

TABLE 41 C. difficile Drug Product Lyophilization Cycle Set PointsTemperature Ramp Soak Step (° C.) (minutes) (minutes) Pressure Loading 5° C. N/A 60 — Freezing 1 −50° C. 183 60 — Annealing −10° C. 133 180Freezing 2 −45° C. 117 90 Vacuum Initiation −45° C. — 60 50 PrimaryDrying −30° C.  75 3420 50 Secondary Drying  30° C. 300 600 50 Storage 5° C.  50 — 50Drug product stability data is summarized in Table 42. The data suggestthat the drug product is physically and chemically stable during storageat 2-8° C. for at least 3 months or at least 1 month at 25° or 40° C.Under both storage conditions, the level of impurities detected by sizeexclusion chromatography (SEC) did not change, nor were there changes inin vitro antigenicity through the latest timepoints tested.

TABLE 42 Stability of Lyophilized Drug Product^(a) Drug ProductFormulation 200 μg/mL mutant toxin A drug substance, 200 μg/mL mutanttoxin B drug substance, 4.5% Trehalose dihydrate, 0.01% Polysorbate 80,10 mM Tris buffer pH 7.4 1 1 3 Test t = 0 Month@25° C. Month@40° C.months @2-8° C. Appearance before White cake essentially White cakeessentially White cake essentially White cake essentiallyReconstitution. free from visible free from visible free from visiblefree from visible foreign particulate foreign particulate foreignparticulate foreign particulate matter matter matter matter Appearanceafter Clear colorless Clear colorless Clear colorless Clear colorlessReconstitution. solution solution solution solution pH 7.5 7.6 7.6 7.5Strength by AEX mutant toxin A drug Mutant toxin A drug Mutant toxin Adrug Mutant toxin A drug (μg/mL) substance 212 substance 193 substance191 substance 193 mutant toxin B drug mutant toxin B drug Mutant toxin Bdrug mutant toxin B drug substance 235 substance 223 substance 222substance 230 Impurity by SEC <2.5% 2.8% 2.8% 2.9% CharacterizationHMMS: 29.6% HMMS: 30.2% HMMS: 30.2% HMMS: 28.5% by SEC Monomer: 68.0%Monomer: 67.1% Monomer: 67.1% Monomer: 68.7% Moisture 0.5 NA NA NA^(a)Lyophilized DP is reconstituted with 60 mM NaCl diluent for thesetests.

Example 40 Vaccine Diluents

For saline, 60 mM NaCl is used as a diluent for the lyophilized drugproduct without any adjuvant to ensure an isotonic solution uponreconstitution.

Alhydrogel:

Alhydrogel “85” 2% (Brenntag) is a commercially available GoodManufacturing Practice (GMP) grade product composed of octahedralcrystalline sheets of aluminum hydroxide. An exemplary Alhydrogeldiluent formulation is shown in Table 43. The exemplary formulation maybe used in combination with the drug product described above.

TABLE 43 Formulation Rationale for Alhydrogel Diluent Component SelectedFormulation dosage form Liquid Suspension Adjuvant dose per 0.5 mL 0.5mg Al pH 6.5 ± 0.5 Buffer 10 mM His Salt 60 mM NaCl Container closures 2mL 13 mm Type 1 Flint Glass Vial, Blowback, West - Flurotec

Studies with the Alhydrogel adjuvant show 100% binding of mutant toxin Adrug substance and mutant toxin B drug substance to 1 mg A1/mLAlhydrogel from pH 6.0 to 7.5. Maximum binding of both drug substanceswas seen at the highest protein concentration tested (300 μg/mL each).

The binding of the proteins to Alhydrogel was also tested with thelyophilized drug product formulation containing 200 μg/mL of each drugsubstance and Alhydrogel ranging from 0.25 to 1.5 mg/ml. The drugproduct was reconstituted with diluents containing the varyingconcentrations of Alhydrogel and the percent of each mutant toxin boundwas measured. All tested concentrations of Alhydrogel demonstrated 100%binding of the antigens.

The binding kinetics of the proteins to Alhydrogel at the target dose ofmutant toxin A drug substance and mutant toxin B drug substance (200μg/mL each) were also assessed. The results show that 100% of the mutanttoxin drug substances were bound to Alhydrogel throughout the 24-hour RTtime course.

CpG 24555 and Alhydrogel:

CpG 24555 is a synthetic 21-mer oligodeoxynucleotide (ODN) having asequence 5-TCG TCG TTTTTC GGT GCT TTT-3 (SEQ ID NO: 48). An exemplaryformulation for a combination of CpG 24555 and Alhydrogel diluents isshown in Table 44. The exemplary formulation may be used in combinationwith the drug product described above.

TABLE 44 Formulation Rationale for CpG/Alhydrogel Diluent ComponentSelected Formulation dosage form Liquid Suspension Adjuvant dose per 0.5mL 0.5 mg Al and 1 mg cpG pH 6.5 ± 0.5 Buffer 10 mM His Salt 60 mM NaClContainer closures 2 mL 13 mm Type 1 Flint Glass Vial, Blowback, West -Flurotec

ISCOMATRIX®:

The ISCOMATRIX® adjuvant is a saponin-based adjuvant known in the art.An exemplary formulation for the ISCOMATRIX® adjuvant formulation isshown in Table 45. The exemplary formulation may be used in combinationwith the drug product described above.

TABLE 45 Formulation Rationale for ISCOMATRIX ® Diluent ComponentSelected Formulation dosage form Liquid Suspension Adjuvant dose per 0.5mL 45units pH 6.2 ± 0.5 Buffer 10 mM phosphate Salt 60 mM NaCl Containerclosures 2 mL 13 mm Type 1 Flint Glass Vial, Blowback, West - Flurotec

Example 41 Immunogenicity of Mutant Toxin Drug Substance CompositionsAdjuvanted with Alhydrogel in NHP Model and Preclinical Proof of Concept

The immunogenicity of mutant toxin A drug substance and mutant toxin Bdrug substance compositions adjuvanted with Alhydrogel in NHPs wasassessed, specifically cynomolgus macaques. NHPs immunized at two-weekintervals (weeks 0, 2, 4) with 10 μg of each mutant toxin A drugsubstance and mutant toxin B drug substance compositions (formulatedwith Alhydrogel) per dose, developed robust neutralizing antitoxinresponses. See Table 46. Both antitoxin A and antitoxin B neutralizingresponses reached a protective range after the third immunization andremained within or above the protective range at least through week 33(last timepoint studied).

Cynomolgus macaques (n=8) were immunized IM at 0, 2 and 4 weeks with 10μg each of mutant toxin A drug substance and mutant toxin B drugsubstance formulated in 250 μg of Alhydrogel. Sera was collected at eachtime point and analyzed in the toxin neutralization assay for functionalantitoxin activity. GMTs are provided in Table 46. The protective titerrange provided in the table depicts the neutralizing antibody titerrange which correlates to significant reduction in recurrence of C.difficile infection in the Merck monoclonal antibody therapy trial.

TABLE 46 Immunogenicity of Mutant Toxin A Drug Substance and MutantToxin B Drug Substance (Formulated in 250 μg Alhydrogel) in CynomolgusMonkeys (50% Neutralization Titer) Week: Wk 0 Wk 1 Wk 2 Wk 3 Wk 4 Wk 5Wk 6 Wk 8 Wk 12 Wk 25 Wk 29 Wk 33 Antitoxin A (Merck/Medarex protectiverange: 666-6,667 for antitoxin A) Titer: 15 19 129 382 336 2469 30692171 1599 1520 1545 2178 Antitoxin B (Merck/Medarex protective range:222-2,222 for antitoxin B) Titer: 10 10 10 10 20 311 410 446 676 16312970 3510Correlation of Human Protective Antibody Titers from Merck mAb TherapyTrial to Titers Induced by Pfizer's Vaccine Candidate in NHPs

The Phase 2 efficacy study with Merck/Medarex mAbs (Lowy et al., N EnglJ Med. 2010 Jan. 21; 362(3):197-205) seemed to demonstrate a correlationbetween the level of neutralizing antitoxin mAbs in the serum and theprevention of recurrence of CDAD. After administration of thetoxin-specific mAbs to humans, serum antibody levels in human recipientsin the range of 10 to 100 μg/mL appear to protect against recurrences(70% reduction in the recurrence of CDAD).

Immunogenic compositions including the mutant toxin drug substances weretested to gauge whether the immunogenic compositions are capable ofinducing a potentially efficacious neutralizing antibody responses inhumans by comparing published data from the Merck/Medarex Phase 2 studyto the levels of antibody induced by the immunogenic compositions in theNHP model. This was accomplished by utilizing previously publishedcharacteristics of the Merck/Medarex mAbs to convert the range of thesemAbs in the serum obtained from subjects that displayed no sign ofrecurrences (10-100 μg/mL) into 50% neutralization titers and comparingthese titers (“protective titer range”) to the titers observed in thepreclinical models described herein. As shown in Table 46, theimmunogenic compositions including the mutant toxin A drug substance andmutant toxin B drug substance adjuvanted with Alhydrogel generatedimmune responses in NHPs that reached the “protective range” after thethird dose and have remained within or above this range through week 33.The level of toxin-neutralizing antibodies induced in NHPs by theinventive C. difficile immunogenic composition is comparable to theserum antibody levels in the Merck/Medarex trial subjects who appearedto be protected from recurrences of CDAD.

Example 42 Immunogenicity of Mutant Toxin Drug Substance CompositionsAdjuvanted with ISCOMATRIX or Alhydrogel/CpG 24555 (Alh/CpG) in NHPModel

In NHPs, both ISCOMATRIX and Alh/CpG statistically significantlyenhanced antitoxin A and B neutralization titers when compared tovaccine administered with Alhydrogel alone (Table 47). Antitoxinresponses above background were elicited at earlier time points byvaccine administered with either Alh/CpG or ISCOMATRIX (week 2-4) ascompared to Alhydrogel alone (week 4-6), which may have an importanteffect on protection from recurrence of CDAD in humans. Compared toAlhydrogel, the immunogenic composition adjuvanted with Alh/CpG or withISCOMATRIX generated antitoxin neutralization titers that reached theprotective range (see also Example 41) more swiftly and that haveremained within or above this range through week 33.

As shown in Table 47, Cynomolgus macaques were immunized IM at weeks 0,2, and 4 with 10 μg each of mutant toxin A drug substance and mutanttoxin B drug substance formulated in 250 μg of Alhydrogel (n=8), or 500μg of CpG+250 μg of Alhydrogel (n=10), or 45 U of ISCOMATRIX (n=10).Sera were collected at each time point and analyzed in the toxinneutralization assay described above for functional antitoxin activity.GMTs are listed in the tables. Asterisks (*) indicate statisticalsignificance (p<0.05) when compared to titers in the Alhydrogel group.The protective titer range represents the neutralizing antibody titerrange which correlates to significant reduction in recurrence of C.difficile infection according to the Merck/Medarex mAb therapy trial.

TABLE 47 Immunogenicity of Adjuvanted Mutant Toxin Drug Substances inNHPs (50% Neutralization Titer) Week: Wk 0 Wk 2 Wk 4 Wk 6 Wk 12 Wk 25 Wk33 Antitoxin A (Merck/Medarex protective range: 666-6,667 for antitoxinA) Alhydrogel 15 129 336 3069 1599 1520 2178 Titer: Alhydrogel + CpG 17*1004 *2162 *15989 *7179 *5049 *7023 Titer: ISCOMATRIX 25 *1283 *3835*19511 *12904 *6992 *7971 Titer: Antitoxin B (Merck/Medarex protectiverange: 222-2,222 for antitoxin B) Alhydrogel 10 10 20 410 676 1631 3510Titer: Alhydrogel + CpG 10 13 *136 *2163 *5076 *9057 *27971 Titer:ISCOMATRIX 10 10 *269 *5325 *9161 *19479 *25119 Titer:

The dose of mutant toxin A drug substance and mutant toxin B drugsubstance administered, in the presence of ISCOMATRIX or Alh/CpGadjuvants, on neutralizing antitoxin antibody titers generated in NHPswas also evaluated. In one study, NHPs were administered a low (10 μg)or a high (100 μg) dose of each mutant toxin drug substance formulatedin ISCOMATRIX. Responses were compared at each time point afterimmunization. As shown in Table 48, antitoxin neutralization titers wererobust in both treatment groups. The antitoxin A titers were nearlyequivalent at most time points between the low dose and high dosegroups, while there was a trend for the antitoxin B titers to be higherin the high dose group.

TABLE 48 Neutralizing Antitoxin Titers in NHPs Following Immunizationwith Either 10 μg or 100 μg of Each of Mutant Toxin Drug Substance andMutant Toxin Drug Substance Administered with ISCOMATRIX (50%Neutralization Titer) Week Wk 0 Wk 2 Wk 3 Wk 4 Wk 6 Wk 8 Wk 12 AntitoxinA (Merck/Medarex protective range: 666-6,667 for antitoxin A)  10 μg 11585 3522 4519 19280 10225 12084 Titer: 100 μg 11 400 1212 2512 994410283 18337 Titer: Antitoxin B (Merck/Medarex protective range:222-2,222 for antitoxin B)  10 μg 10 10 112 266 3710 2666 7060 Titer:100 μg 10 10 303 469 6016 4743 20683 Titer:

As shown in Table 48, Cynomolgus macaques (n=5) were immunized IM atweeks 0, 2, and 4 with 10 μg or 100 μg each of mutant toxin A drugsubstance and mutant toxin B drug substance formulated with 45U ofISCOMATRIX. Sera were collected at each time point and analyzed in thetoxin neutralization assay for functional antitoxin activity. GMTs arelisted in the table. The protective titer range represents theneutralizing antibody titer range which correlates to significantreduction in recurrence of C. difficile infection in the Merck/MedarexmAb therapy trial.

In an effort to enhance the kinetics of antitoxin B responses, NHPs wereimmunized with a constant dose of mutant toxin A drug substance (10 μg)that was mixed with an increasing dose of mutant toxin B drug substance(10, 50, or 100 μg) in the presence of ISCOMATRIX or Alh/CpG adjuvants.Regardless of adjuvant, there was a trend for groups that receivedhigher doses of mutant toxin B drug substance (either 50 or 100 μg) toinduce higher antitoxin B neutralizing responses in comparison to the 10μg dose of mutant toxin B drug (Table 50, marked by * to indicatestatistically significant increases). This trend was observed at mosttime points after the final immunization. However, in some cases,antitoxin A neutralizing responses showed a statistically significantdecrease (marked by A in Table 49) when the amount of mutant toxin B wasincreased.

As shown in Table 49 and Table 50, NHPs (10 per group) were immunized IMat weeks 0, 2, and 4 with different ratios of mutant toxin A drugsubstance and mutant toxin B drug substance (10 μg of mutant toxin Adrug substance plus either 10, 50, or 100 μg of mutant toxin B drugsubstance; designated 10A:10B, 10A:50B and 10A:100B, respectively, inTable 49 and Table 50), formulated with ISCOMATRIX (45U per dose) orwith Alh/CpG/(250 μg/500 μg per dose). Table 49 shows Antitoxin Atiters. Table 50 shows Antitoxin B titers. GMTs are listed in thetables. The protective titer range represents the neutralizing antibodytiter range which correlates to significant reduction in recurrence ofC. difficile infection in the Merck mAb therapy trial. The symbol A,represents statistically significant decrease in neutralizing titers(p<0.05) compared to the 10A: 10B group. The asterisk symbol, *,represents statistically significant increase in neutralizing titers(p<0.05) compared to the 10A: 10B group.

TABLE 49 Neutralizing Antitoxin Titers in NHPs Following Immunizationwith 10 μg Mutant Toxin A Drug Substance Combined with 10, 50, or 100 μgMutant Toxin B Drug Substance using ISCOMATRIX or Alh/CpG as Adjuvants(50% Neutralization Titer) Week: Wk 0 Wk 2 Wk 4 Wk 6 Wk 12 Wk 25 Wk 33Antitoxin A (Merck/Medarex protective range: 666-6,667 for antitoxin A)ISCOMATRIX 10A:10B 25 1283 3835 19511 12904 6992 7971 Titer: 10A:50B 29906 2917 16126 {circumflex over ( )}7756 {circumflex over ( )}4208 5965Titer: 10A:100B 20 982 2310 {circumflex over ( )}5034 {circumflex over( )}5469 {circumflex over ( )}4007 3780 Titer: Antitoxin A(Merck/Medarex protective range: 666-6,667 for antitoxin A) Alh/CpG10A:10B 17 1004 2162 15989 7179 5049 7023 Titer: 10A:50B 20 460 172816600 6693 6173 8074 Titer: 10A:100B 27 {circumflex over ( )}415 159513601 6465 5039 6153 Titer:

TABLE 50 Neutralizing Antitoxin Titers in NHPs Following Immunizationwith 10 μg Mutant Toxin A Drug Substance Combined with 10, 50, or 100 μgMutant Toxin B Drug Substance using ISCOMATRIX or Alh/CpG as Adjuvants(50% Neutralization Titer) Week: Wk 0 Wk 2 Wk 4 Wk 6 Wk 12 Wk 25 Wk 33Antitoxin B (Merck/Medarex protective range: 222-2,222 for antitoxin B)ISCO- 10 10 269 5325 9161 19479 25119 MATRIX Titer: Titer: 13 *20 *6044861 10801 20186 *57565 Titer: 10 *23 *862 *10658 10639 *33725 *56073Antitoxin B (Merck/Medarex protective range: 222-2,222 for antitoxin B)Alh/CpG 10 13 136 2163 5076 9057 27971 Titer: Titer: 10 15 *450 *5542*9843 15112 50316 Titer: 11 17 *775 *13533 *11708 *17487 26600

Example 43 Five-Week Repeat-Dose IM Toxicity Study with an ImmunogenicComposition in Cynomolgus Monkeys, with a 4-Week Recovery Period

The 5-week IM repeat-dose toxicity study with PF-06425095 (animmunogenic composition including triple mutant toxin A drug substanceand triple mutant toxin B drug substance in a combination with adjuvantsaluminum hydroxide and CpG 24555) in Cynomolgus monkeys was conducted toassess the potential toxicity and immunogenicity of C. difficile triplemutant toxin A drug substance and triple mutant toxin B drug substancein a combination with the adjuvants aluminum hydroxide and CpG 24555(PF-06425095). PF-06425095 at 0.2 or 0.4 mg/dose triple mutant toxin Adrug substance and triple mutant toxin B drug substance (low- andhigh-dose immunogenic composition groups, respectively), 0.5 mg aluminumas aluminum hydroxide, and 1 mg CpG 24555 and the adjuvant combinationalone (aluminum hydroxide+CpG 24555; PF-06376915) were administered IMto cynomolgus monkeys (6/sex/group) as a prime dose followed by 3booster doses (Days 1, 8, 22, and 36). A separate group of animals(6/sex) received 0.9% isotonic saline at an approximate pH of 7.0. Theimmunogenic composition vehicle was composed of 10 mM Tris buffer at pH7.4, 4.5% trehalose dihydrate, and 0.1% polysorbate 80. The adjuvantcontrol vehicle was composed of 10 mM histidine buffer with 60 nM NaClat pH 6.5. The total dose volume was 0.5 mL per injection. All doseswere administered into the left and/or right quardriceps muscle.Selected animals underwent a 4-week dose-free observation period toassess for reversibility of any effects observed during the dosing phaseof the study.

There were no adverse findings in this study. PF-06425095 waswell-tolerated and produced only local inflammatory reaction withoutevidence of systemic toxicity. During the dosing phase, dose-dependentincreases from pretest in fibrinogen (23.1% to 2.3×) on Days 4 and 38and C-reactive protein on Days 4 (2.1× to 27.5×) and 38 (2.3× to101.5×), and globulin (11.1% to 24.1%) on Day 36 and/or 38, were seen inimmunogenic composition-treated groups and were consistent with theexpected inflammatory response to administration of an adjuvantedimmunogenic composition.

The increases in fibrinogen and C-reactive protein noted on Day 4 hadpartially recovered by Day 8 with increases in fibrinogen (25.6% to65.5%) and C-reactive protein (4.5× and 5.6×) in the high-doseimmunogenic composition group only. Increases in interleukin (IL)-6 wereobserved in the low- and high-dose immunogenic composition groups on Day1, Hour 3 (8.3× to 127.2× individual values Day 1, Hour 0, doseresponsive) and Day 36, Hour 3 (9.4× to 39.5× individual values Day 36,Hour 0). There were no changes observed in the other cytokines (IL-10,IL-12, Interferon-Inducible Protein (IP-10), and Tumor Necrosis Factor α(TNF-α). Increases in these acute phase proteins and cytokine were partof the expected normal physiologic response to the administration offoreign antigen. There were no PF 06425095-related or adjuvant-relatedalterations in these clinical pathology parameters in the recovery phase(cytokines were not evaluated during the recovery phase). In addition,there were localized changes at the injection sites, which were ofsimilar incidence and severity in the adjuvant control group and thelow- and high-dose immunogenic composition groups; hence, they were notdirectly related to PF-06425095. During the dosing phase, the changesincluded minimal to moderate chronic-active inflammation that wascharacterized by separation of muscle fibers by infiltrates ofmacrophages, which often contained basophilic granular material(interpreted as aluminum-containing adjuvant), lymphocytes, plasmacells, neutrophils, eosinophils, necrotic debris, and edema. Thebasophilic granular material was also present extracellularly withinthese foci of chronic-active inflammation. At the end of the recoveryphase, there was minimal to moderate chronic inflammation andmononuclear cell infiltrate, and minimal fibrosis. These injection sitefindings represent a local inflammatory response to the adjuvant. Othermicroscopic changes included minimal to moderate increased lymphoidcellularity in the iliac (draining) lymph node and minimal increasedcellularity in germinal centers in the spleen that were noted during thedosing phase in the adjuvant control group and the low- and high-doseimmunogenic composition groups. At the end of the recovery phase, thesemicroscopic findings were of lower severity. These effects represent animmunologic response to antigenic stimulation, and were a pharmacologicresponse to the adjuvant or PF-06425095. There was no testarticle-related increase in anti-DNA antibodies.

Based on absence of adverse findings, the no observed adverse effectlevel (NOAEL) in this study is the high-dose immunogenic compositiongroup (0.4 mg of triple mutant toxin A drug substance and triple mutanttoxin B drug substance/dose as PF-06425095) administered as two 0.5 mLinjections for four doses.

Example 44 Efficacy of Seropositive NHP Sera Passively Transferred toHamsters

Groups of 5 Syrian golden hamsters were administered an oral dose ofclindamycin antibiotic (30 mg/kg) to disrupt normal intestinal flora.After five days, the hamsters were challenged with an oral dose of wildtype C. difficile spores (630 strain, 100 cfu per animal), andadministered intraperitoneally (IP) with NHP sera according to Table 51.Without being bound by mechanism or theory, disease symptoms followingchallenge with the spores typically manifest beginning about 30-48 hourspost-challenge.

The NHP sera that were administered to the hamsters were pooled from NHPserum samples exhibiting the highest titer (anti-toxin A sera andanti-toxin B sera) following three immunizations with mutant toxin Adrug substance and mutant toxin B drug substance (10:10, 10:50, and10:100 A:B ratios), formulated with ISCOMATRIX (see Example 42, Table49, and Table 50). The NHP sera were collected from timepoints at weeks5, 6, and 8 (immunizations occurred at weeks 0, 2, and 4), as describedin Examine 42. Results are shown in Tables 52-54 below. The symbol “+”indicates a Geometric mean (GM) in ( ) that does not include animal #3,non-responder. “*TB” represents terminal bleed, the day the animal waseuthanized, which is not the same for all animals.

TABLE 51 Experimental design Administered No. Group composition animalsRoute Schedule 1 Seropositive NHP sera 5 IP Challenge Day 0 “1(unconcentrated) Dose day 0 dose” Bleed days 0, 1, 2, TB on day 11 2Seropositive NHP sera 5 IP Challenge Day 0 “2 (unconcentrated) Dose days0, 1 dose” Bleed days 0, 1, 2, TB on day 11

TABLE 52 Anti-toxin A Neutralization Titers in Hamster Sera Following 1or 2 IP doses of NHP Sera (50% Neutralization Titer in RLU) Day Hamster1 Hamster 2 Hamster 3 Hamster 4 Hamster 5 GM SE 1 D0 50 50 50 50 50  500 dose D1 2877 4008 2617 4917 1872 3081 538 D2 1983 3009 2750 2902 11172214 357 TB* 3239 537 155 977 972  762 538 (d4) (d9) (d11) (d9)  (d2) 2D0 50 50 50 50 50  50 0 dose D1 1154 2819 50 429 1174  606 475  (1131)⁺D2 4119 4674 1899 545 2113 862 TB* 1236 1267 1493 50 1877  738 306 (d9)(d8) (d4)  (d11) (d9) Input NHP sera = 41976

TABLE 53 Anti-toxin B Neutralization Titers in Hamster Sera Following 1or 2 IP doses of NHP Sera (50% Neutralization Titer in RLU) Day Hamster1 Hamster 2 Hamster 3 Hamster 4 Hamster 5 GM SE 1 D0 50 50 50 50 50  500 dose D1 1846 4254 1347 5178 406 1859 904 D2 992 1795 2585 2459 11451669 327 TB* 1744 50 50 265 544  229 317 (d4) (d9) (d11) (d9)  (d2) 2 D050 50 50 50 50  50 0 dose D1 1189 2229 50 550 3920  778 687  (1546)⁺ D22288 2706 1452 287 1268 477 TB* 301 694 682 50 1334  394 217 (d9) (d8)(d4)  (d11) (d9) Input NHP sera = 23633

TABLE 54 Percentage of hamsters protected from severe CDAD following 1or 2 IP doses of NHP sera Days post-infection 0 2 4 6 8 10 11 1 dose NHPSera 100% 80% 60% 60% 60% 20% 20% 2 dose NHP Sera 100% 100% 80% 80% 60%20% 20% Placebo 100% 75% 50% 25% 0% n/a n/a

In another study, Syrian golden hamsters were administered an oral doseof clindamycin antibiotic (30 mg/kg) to disrupt normal intestinal flora.After five days, the hamsters were challenged with an oral dose of wildtype C. difficile spores (630 strain, 100 cfu per animal), andadministered intraperitoneally (IP) NHP sera according to Table 55.Without being bound by mechanism or theory, disease symptoms followingchallenge with the spores typically manifest beginning about 30-48 hourspost-challenge.

The NHP sera that were administered to the hamsters were pooled fromsamples collected from NHPs following three immunizations with mutanttoxin A drug substance and mutant toxin B drug substance (10:10, 10:50,and 10:100 A:B ratios), formulated with Alhydrogel and CpG 24555 (seeExample 42, Table 49, and Table 50). The NHP sera were collected fromtimepoints at weeks 5, 6, 8, and 12 as described in Examine 42 (NHPswere immunized on weeks 0, 2, and 4). Results are shown in Tables 56-59below. Sera from the hamsters were further investigated to determineinhibitory concentration (IC₅₀) value, which were determined using thetoxin neutralization assay described above. The level oftoxin-neutralizing antibodies induced in hamsters by the inventive C.difficile immunogenic composition is comparable to the serum antibodylevels in the Merck/Medarex trial subjects who appeared to be protectedfrom recurrences of CDAD.

TABLE 55 Experimental Design Administered Group Composition No. RouteSchedule 1 Seropositive NHP 5 IP Challenge D 0 sera Dose D 0, 1, 3, 5, 72 Seropositive NHP 5 IP no challenge sera Dose D 0, 1, 3, 5, 7, 3Seropositive NHP 10 IP Challenge D 0 sera Dose D 0, 1, 3, 5, 7 4 Placebo5 IM Challenge D 0

TABLE 56 Anti-toxin A Neutralization Titers^(a) in Hamster SeraFollowing 1 or 2 IP doses of NHP Sera (50% Neutralization Titer in RLU)Challenged Not Challenged Day (Groups 1 and 3) (Group 2) p Value 0 11 120.5933 1 380 720 0.034* 3 666 1220 0.0256* 5 864 1367 0.0391* 7 564 16880.0411* 11 263 1281 0.001* Input NHP sera pool = 9680 ^(a)titersexpressed as geometric means for each group (n = 15 at day 0 for“challenged” group, n = 5 for “not challenged” group) Merck/Medarexprotective range: 666-6,667 for antitoxin A The asterisk “*” indicates asignificant difference.

TABLE 57 Anti-toxin B Neutralization Titers^(a) in Hamster SeraFollowing 1 or 2 IP doses of NHP Sera (50% Neutralization Titer in RLU)Challenged Not Challenged Day (Groups 1 and 3) (Group 2) p Value 0 10 100.3343 1 465 828 0.0579 3 765 1400 0.0273* 5 941 1734 0.0226* 7 611 18770.0498* 11 194 1436 0.0047* Input NHP sera pool = 19631 ^(a)titersexpressed as geometric means for each group (n = 15 at day 0 for“challenged” group, n = 5 for “not challenged” group) Merck/Medarexprotective range: 222-2,222 for antitoxin B The asterisk “*” indicates asignificant difference.

TABLE 58 Percentage of hamsters protected from severe CDAD following IPdose of NHP sera Days post-infection 0 2 4 6 8 10 11 Groups 1 and 3 100%73% 53% 53% 47% 33% 33% Placebo (Group 2) 100% 50% 0%

TABLE 59 IC₅₀ values from Toxin-specific 50% Neutralization Titers IC₅₀of Anti Toxin A IC₅₀ of Anti Toxin B Day of Post Dose Day of Post DoseAnimal ID 0 1 3 5 7 11 Animal ID 0 1 3 5 7 11 Challenged 1-1 10 50 338died 1-1 10 50 254 died D4 D4 1-2 10 614 579 777 605 192 1-2 10 720 659896 475 157 1-3 10 710 1035  845 548 Died 1-3 10 867 1017  988 694 D101-4 10 850 588 942 1116 296 1-4 10 1158 555 1158  1806 250 1-5 10 780 895* 1-5 10 910  687* 3-1 10 647 Died 3-1 10 598 Died D2 D2 3-2 10 331Died 3-2 10 290 Died D2 D2 3-3 10 660 1273  849 692 640 3-3 10 717 1623 870 791 574 3-4 10 536 493 1102 1314 Died 3-4 10 618 598 977 1478 DiedD9 D9 3-5 10 817 807 774 1077 187 3-5 10 772 1260  850 913 243 3-6 10117 649 803 50 186 3-6 10 1038 773 883 50 50 3-7 10 50 Died 3-7 10 50Died D2 D2 3-8 10 149 659 650* 3-8 10 121 1010   517* 3-9 30 797 1170*3-9 10 1008 1720*  3-10 10 792 Died  3-10 10 835 Died D2 D2 GeoMean 11380 666 864 564 263 GeoMean 10 465 765 941 611 194 Not Std Error 1 78 86 41 163 88 Std Error 0 94 125  38 224 88 Challenged 2-1 10 697 1634 1597 2219 1709 2-1 10 890 1777  1910  3229 1355 2-2 10 779 1207  13221755 1327 2-2 10 939 1378  1564  1897 1379 2-3 10 581 669 722 1401 11182-3 10 828 837 865 1484 1404 2-4 26 856 1540  1875 1830 1826 2-4 10 7481780  2939  1880 2650 2-5 10 715 1331  1668 1374 744 2-5 10 752 1475 2064  1364 880 GeoMean 12 720 1220  1367 1688 1281 GeoMean 10 828 1400 1734  1877 1436 Std Error 3 46 169 199 156 197 Std Error 0 38 173 338332 296 *= deceased on that day

Example 45 Characterization of Mutant Toxin Drug Substances

The primary structure of triple mutant toxin A is shown in SEQ ID NO: 4.The NH₂-terminal Met residue at position 1 of SEQ ID NO: 4 is originatedfrom the initiation codon of SEQ ID NO: 12 and is absent in isolatedprotein (e.g., see SEQ ID NO: 84). Accordingly, in Example 12 to Example45, “SEQ ID NO: 4” refers to SEQ ID NO: 4 wherein the initial methionine(at position 1) is absent. Both purified triple mutant toxin A (SEQ IDNO: 4) (Drug Substance Intermediate—Lot L44993-132) and EDC/NHS treatedtriple mutant toxin A (SEQ ID NO: 4)(“mutant toxin A Drug Substance”—LotL44898-012) displayed a single NH₂-terminal sequence starting atSLISKEELIKLAYSI (positions 2-16 of SEQ ID NO: 4).

The primary structure of triple mutant toxin B is shown in SEQ ID NO: 6.The NH₂-terminal Met residue at position 1 of SEQ ID NO: 6 isoriginating from the initiation codon and is absent in isolated protein(e.g., see SEQ ID NO: 86). Accordingly, in Example 12 to Example 45,“SEQ ID NO: 6” refers to SEQ ID NO: 6 wherein the initial methionine (atposition 1) is absent. Both purified triple mutant toxin B (SEQ ID NO:6) (Drug Substance Intermediate—Lot 010) and EDC/NHS treated triplemutant toxin B (SEQ ID NO: 6)(“mutant toxin B Drug Substance”—LotL44906-153) displayed a single NH₂-terminal sequence starting atSLVNRKQLEKMANVR (positions 2-16 of SEQ ID NO: 6).

Circular dichroism (CD) spectroscopy was used to assess secondary andtertiary structure of triple mutant A (SEQ ID NO: 4) and mutant toxin Adrug substance. CD spectroscopy was also used to assess secondary andtertiary structure of the triple mutant toxin B (SEQ ID NO: 6) and themutant toxin B drug substance. CD spectroscopy was also used to assesspotential effects of pH on structure. The effect of EDC treatment ontriple mutant toxin A was analyzed by comparing CD data obtained formutant toxin A drug substance to the data obtained for triple mutanttoxin A. The effects of EDC treatment on triple mutant toxin B (SEQ IDNO: 6) were analyzed by comparing CD data obtained for mutant toxin Bdrug substance to the data obtained for triple mutant toxin B.

Mutant toxin A drug substance far-UV CD data were obtained at variouspH. Spectra recorded at pH 5.0-7.0 are indicative of high proportion ofα-helices in the secondary structure, suggesting that polypeptidebackbone of the protein adopts well-defined conformation dominated byα-helices.

Near-UV CD spectra of mutant toxin A drug substance were also obtained.Strong negative ellipticity between 260 and 300 nm is an indication thataromatic side chains are in the unique rigid environment, i.e. mutanttoxin A drug substance possesses tertiary structure. In fact,characteristic features arising from individual types of aromatic sidechains can be distinguished within the spectrum: shoulder at ˜290 nm andlargest negative peak at ˜283 nm are due to absorbance of the polarizedlight by ordered tryptophan side chains, negative peak at 276 nm is fromthe tyrosine side chains, and minor shoulders at 262 and 268 nm areindicative of the phenylalanine residues participating in tertiarycontacts. Far- and near-UV results provide evidence that mutant toxin Adrug substance retains compactly folded structure at physiological pH.Nearly identical far- and near-UV CD spectra observed at pH 5.0-7.0indicate that no detectable structural changes are taking place withinthis pH range. CD data could not be collected at pH 3.0 and 4.0, sincethe protein was insoluble at these pH points. In comparing far- andnear-UV CD spectra of mutant toxin A drug substance with those of thetriple mutant toxin A, spectra of both proteins are essentiallyidentical under all of the experimental conditions studied, indicatingthat EDC treatment had no detectable effects on secondary and tertiarystructure of the triple mutant toxin A. This finding is in agreementwith the gel-filtration and analytical ultracentrifugation results,which show no detectable changes in Stokes radii andsedimentation/frictional coefficients, respectively.

Mutant toxin A drug substance (as well as triple mutant toxin A)contains 25 tryptophan residues that are spread throughout the primarysequence and can serve as convenient intrinsic fluorescence probes.Fluorescence emission spectra of mutant toxin A drug substance between300 and 400 nm as a function of temperature were obtained. At 6.8° C.mutant toxin A drug substance shows characteristic tryptophanfluorescence emission spectrum upon excitation at 280 nm. Fluorescenceemission maximum is observed at ˜335 nm, indicating that tryptophanresidues are in non-polar environment, typical of protein interiorsrather than of polar aqueous environments. The fluorescence emissionspectra results, together with the results of the CD experimentspresented in this report, confirm that mutant toxin A drug substanceretains compact folded structure.

Fluorescence of the extrinsic probe 8-anilino-1-naphtalene sulfonic acid(ANS) was used to characterize possible conformational changes in mutanttoxin A drug substance and triple mutant toxin A upon changes in pH. Ascan be seen from the results, there is essentially no increase in ANSfluorescence intensity when either mutant toxin A drug substance ortriple mutant toxin A are titrated with the probe at pH 7.0, suggestingthat no hydrophobic surfaces are exposed on the proteins under theseconditions. Shifting pH to 2.6 leads to a dramatic increase in ANSfluorescence quantum yield upon increase in probe's concentration, untilfluorescence quantum yield reaches apparent saturation. This increase inANS fluorescence quantum yield indicates that at low pH (2.6), bothmutant toxin A drug substance and triple mutant toxin A undergopH-induced conformational change that exposes hydrophobic surfaces. Suchconformational changes indicate that EDC-induced modification andinactivation of triple mutant toxin A did not restrict conformationalplasticity of mutant toxin A drug substance (DS).

Effect of EDC treatment on hydrodynamic properties of triple mutanttoxin A was evaluated using size-exclusion chromatography on a G4000SWXL column. Mutant toxin A drug substance and triple mutant toxin Awere injected onto the G4000 SWXL column equilibrated at pH 7.0, 6.0,and 5.0. The data indicate that no differences in the Stoke's radius ofmutant toxin A drug substance and triple mutant toxin A can be detectedusing size exclusion chromatography. Therefore, EDC treatment has notdramatically affected hydrodynamic properties and, correspondingly,overall molecular shape of the triple mutant toxin A.

Further analysis of triple mutant toxin A and mutant toxin A drugsubstance was performed using multi-angle laser light scattering (MALLS)technique. Treatment of triple mutant toxin A with EDC resulted ingeneration of heterogeneous mixture composed of various multimeric andmonomeric species. Such heterogeneity reflects introduction of a largenumber of EDC-induced inter- and intra-molecular covalent bonds betweencarboxyls and primary amines of the protein.

Obtained data provide physical and chemical characteristics of triplemutant toxin A and mutant toxin A drug substance (triple mutant toxin Atreated with EDC) and describe the key features of their primary,secondary, and tertiary structure. Generated data demonstrate thattreatment of triple mutant toxin A with EDC resulted in covalentmodification of its polypeptide chain but did not affect secondary andtertiary structures of the protein. Treatment with EDC leads to intra-and intermolecular cross-linking. The biochemical and biophysicalparameters obtained for mutant toxin A drug substance (as well as triplemutant toxin A) are presented in Table 60.

TABLE 60Major Biochemical and Biophysical Parameters Obtained for TripleMutant Toxin A (SEQ ID NO: 4) and Mutant Toxin A Drug SubstanceTriple Mutant toxin A Mutant Toxin A Drug Parameter (SEQ ID NO: 4)Substance Number of amino acid 2709 2709 residues N-terminal sequenceSLISKEELIKLAYSI SLISKEELIKLAYSI (positions 2-16 (positions 2-16of SEQ ID NO: 4) of SEQ ID NO: 4) Mol mass (from AA 308 kDa 308 kDasequence) Mol mass (from SEC- 299 kDa 300 kDa and MALLS) 718-1139 kDaExtinction coefficient 1.292 or 1.292 or at 280 nm 1.275 (mg/ml)⁻¹cm⁻¹1.275 275 (mg/ml)⁻¹cm⁻¹ Theoretical pl 5.57 ND Partial specific mol0.735 cm³/g 0.735 cm³/g volume at 20° C. Anhydrous volume/monomer 3.8 ×10⁻¹⁹cm³ 3.8 × 10⁻¹⁹cm³ Sedimentation 9.2 S 9.2 S coefficient/monomerFrictional coefficient 1.69 1.69 ratio (f/f₀) Stokes radius/monomer78.4 ± 1.1 77.9 Fluorescence max (λex = 334-335 nm 334-335 nm 280 nm)Near-UV CD spectrum 284 nm and 278 nm 284 nm and 278 nm minimaMean res ellipticity at −138 ± 7 & −130 ± 7 −138 ± 8 & 284 & 278 nm−131 ± 10 Mean res ellipticity −8989 ± 277 −7950 ± 230 at 222 nmDSC unfolding transitions 47.3° C. and 47.9° C. and maxima (PBS, pH 7.4)53.6° C. 54.1° C.

Mutant toxin B drug substance far-UV CD data were obtained at variouspH. Spectra recorded at pH 5.0-7.0 are indicative of high proportion ofα-helices in the secondary structure, suggesting that polypeptidebackbone of the protein adopts well-defined conformation dominated byα-helices.

Near-UV CD spectra of mutant toxin B drug substance were also obtained.Strong negative ellipticity between 260 and 300 nm is an indication thataromatic side chains are in the unique rigid environment, i.e. mutanttoxin B drug substance possesses tertiary structure. In fact,characteristic features arising from individual types of aromatic sidechains can be distinguished within the spectrum: shoulder at ˜290 nm andlargest negative peak at ˜283 nm are due to absorbance of the polarizedlight by ordered tryptophan side chains, negative peak at 276 nm is fromthe tyrosine side chains, and minor shoulders at 262 and 268 nm areindicative of the phenylalanine residues participating in tertiarycontacts. Far- and near-UV CD spectra provide evidence that mutant toxinB drug substance retains compactly folded structure at physiological pH.Very similar far- and near-UV CD spectra observed at pH 5.0-7.0 indicatethat no detectable secondary or tertiary structural changes are takingplace within this pH range. CD data could not be collected at pH 3.0 and4.0, since the protein was insoluble at these pH points.

In comparing far- and near-UV CD spectra of mutant toxin B drugsubstance with those of the triple mutant toxin B, spectra of bothproteins are very similar between pH 5.0 and 7.0, indicating that EDCtreatment had no detectable effects on secondary and tertiary structureof the protein.

Triple mutant toxin B contains 16 tryptophan residues that are spreadthroughout the primary sequence and can serve as convenient intrinsicfluorescence probes. Fluorescence emission spectra of mutant toxin Bdrug substance between 300 and 400 nm as a function of temperature wereobtained. At 7° C. mutant toxin B drug substance shows characteristictryptophan fluorescence emission spectrum upon excitation at 280 nm.Fluorescence emission maximum is observed at ˜335 nm, indicating thattryptophan residues are in non-polar environment, typical of proteininteriors rather than of polar aqueous environments. This result,together with the results of the CD experiments (see above), confirmthat mutant toxin B drug substance retains compact folded structure.

Fluorescence of the extrinsic probe 8-anilino-1-naphtalene sulfonic acid(ANS) was used to characterize possible conformational changes in mutanttoxin B drug substance and triple mutant toxin B upon changes in pH. Ascan be seen from the results, there is essentially no increase in ANSfluorescence intensity when either mutant toxin B drug substance ortriple mutant toxin B are titrated with the probe at pH 7.0, suggestingthat no hydrophobic surfaces are exposed on the proteins under theseconditions. Shifting pH to 2.6 leads to a dramatic increase in ANSfluorescence quantum yield upon increase in probe's concentration in thepresence of mutant toxin B drug substance, until fluorescence quantumyield reaches apparent saturation. This increase in ANS fluorescencequantum yield indicates that at low pH (2.6), mutant toxin B drugsubstance undergoes pH-induced conformational change that exposeshydrophobic surfaces. Such conformational changes indicate thatEDC-induced modification and inactivation of triple mutant toxin B didnot restrict conformational plasticity of mutant toxin B drug substance(DS).

Effect of EDC treatment on hydrodynamic properties of triple mutanttoxin B was evaluated using size-exclusion chromatography on a G4000SWXL column. mutant toxin B drug substance and triple mutant toxin Bwere injected onto the G4000 SWXL column equilibrated at pH 7.0, 6.0,5.0. The data indicate that no differences in the Stoke's radius ofmutant toxin B drug substance and triple mutant toxin B can be detectedusing size-exclusion chromatography, therefore EDC treatment has notdramatically affected hydrodynamic properties and, correspondingly,overall molecular shape of the protein.

Further analysis of triple mutant toxin B and mutant toxin B drugsubstance was performed using multi-angle laser light scattering (MALLS)technique. Treatment of triple mutant toxin B with EDC resulted ingeneration of more heterogeneous mixture that is composed of variousmultimeric and monomeric species. Such heterogeneity reflectsintroduction of a large number of EDC-induced inter- and intra-molecularcovalent bonds between carboxyls and primary amines of the protein.

Obtained data provide physical and chemical characteristics of triplemutant toxin B and mutant toxin B drug substance (triple mutant toxin Btreated with EDC) and describe the key features of their primary,secondary, and tertiary structure. Generated data demonstrate thattreatment of triple mutant toxin B with EDC resulted in covalentmodification of its polypeptide chain but did not affect secondary andtertiary structures of the protein. Treatment with EDC leads to intra-and intermolecular cross-linking. The major biochemical and biophysicalparameters obtained for mutant toxin B drug substance (as well as triplemutant toxin B) are presented in Table 61.

TABLE 61Major Biochemical and Biophysical Parameters Obtained for TripleMutant Toxin B (SEQ ID NO: 6) and Mutant Toxin B Drug SubstanceTriple mutant toxin B Mutant Toxin B Drug Parameter (SEQ ID NO: 6)Substance Number of amino acid 2365 2365 residues N-terminal sequenceSLVNRKQLEKMANVR SLVNRKQLEKMANVR (positions 2-16 of (positions 2-16 ofSEQ ID NO: 6) SEQ ID NO: 6) Mol mass (from AA 269.5 kDa 269.5 kDasequence) Mol mass (from SEC- 255 kDa and ~1,754 kDa 264, 268, 706,MALLS) and 2,211 kDa Extinction coefficient 1.067 (mg/ml)⁻¹cm⁻¹1.067 (mg/ml)⁻¹cm⁻¹ at  280 nm Theoretical pl 4.29 NDPartial specific mol 0.734 cm³/g 0.734 cm³/g volume at 20° C. Anhydrous3.3 × 10⁻¹⁹cm³ 3.3 × 10⁻¹⁹cm³ volume/monomer Sedimentation 9.1 ± 0.2 S9.4 S coefficient/monomer Frictional coefficient 1.58 ± 0.03 1.53ratio (f/f₀) Stokes radius /monomer 76.2 76.2 Fluorescence max (λex =335 nm 335 nm 280 nm) Near-UV CD negative 290, 283, 276, 290, 283, 276,bands 268, 262 nm 268, 262 nm Far-UV CD negative 208 and 222 nm208 and 222 nm bands DSC unfolding transition 48.8 ± 0.0° C. and 48.2 ±0.3° C. and midpoints 52.0 ± 0.1° C. 54.3 ± 0.2° C.T_(m1) and T_(m2) (PBS, pH 7.0)

Example 46 Perfusion Fermentation for Toxoid B Triple Mutant

Toxoid B (triple mutant) seed cultures: inoculated each 1 L bottlecontaining 400 mL of medium with 1 ml seed, incubate at 37° C.,stationary overnight (˜15 hrs). The final OD₆₀₀ should be 3.0-4.0.Working vol: 3 L (2.7 L medium+300 mL inoculum). Each fermenter had 1Rushton impeller and a tube sparger. Initial conditions: Temperature:37° C., N2 flow: ˜0.5 vvm, sparged. Controllers: pH controlled at 7.0with 5N NaOH. Foam controlled by automatic addition of PPG-2000, with0.25 mL/L added to the fermenter medium before sterilization.

Perfusion culture of C. difficile was performed using a stack of 2SARTOCON Slice Cassettes, 0.2 μm pore size of HYDROSART filter material,0.1 square meter surface area per cassette. The 3 L was added to thefermentors in the 10 L glass fermentors. The perfusion started when theOD reaches the target ˜4 for 2 hour intervals of increasing speed as0.75 L/hr, 1.5 L/hr, 2.25 L/hr, 3 L/hr for example 1, FIG. 28. Theperfusion will be started with fermentation medium when the OD reachesthe target ˜4 for 2 hour intervals of increasing speed as 0.75 L/hr, 1.5L/hr, 3 L/hr, and 6 L/hr for example 2, FIG. 29. At the start signal forperfusion, the recirculation pump was started up at the desired speed at1.3 L/min crossflow.

Example 1, FIG. 28: Final OD 50 and toxoid B titer 243 mg/L wereobtained.

Example 2, FIG. 29: Final OD 59 and and toxoid B titer 306 mg/L wereobtained.

The invention also provides the following embodiments as defined in theclauses below:

-   Clause 1. An isolated polypeptide comprising SEQ ID NO: 183.-   Clause 2. An isolated polypeptide comprising SEQ ID NO: 184.-   Clause 3. An immunogenic composition comprising an isolated    polypeptide comprising SEQ ID NO: 183.-   Clause 4. An immunogenic composition comprising an isolated    polypeptide comprising SEQ ID NO: 184.-   Clause 5. A culture medium comprising soy hydrolysate, yeast    extract, and glucose, and wherein the medium comprises a Clostridium    difficile bacterium derived from VPI 11186, wherein the bacterium    lacks an endogenous polynucleotide encoding a toxin, wherein the    bacterium comprises a polynucleotide encoding a mutant C. difficile    toxin, wherein the bacterium further comprises a Clostridium    sporogenes feredoxin (fdx) promoter.-   Clause 6. A method of culturing Clostridium difficile comprising    culturing C. difficile in a medium, wherein the medium comprises soy    hydrolysate, yeast extract, and glucose, and wherein the medium    comprises a Clostridium difficile bacterium derived from VPI 11186,    wherein the bacterium lacks an endogenous polynucleotide encoding a    toxin, wherein the bacterium comprises a polynucleotide encoding a    mutant C. difficile toxin, wherein the bacterium further comprises a    Clostridium sporogenes feredoxin (fdx) promoter.-   Clause 7. A method of producing a Clostridium difficile toxin    comprising culturing C. difficile in a medium under suitable    conditions to produce a toxin, and isolating the toxin from the    medium; wherein the medium comprises soy hydrolysate, yeast extract,    and glucose, and wherein the medium comprises a Clostridium    difficile bacterium derived from VPI 11186, wherein the bacterium    lacks an endogenous polynucleotide encoding a toxin, wherein the    bacterium comprises a polynucleotide encoding a mutant C. difficile    toxin, wherein the bacterium further comprises a Clostridium    sporogenes feredoxin (fdx) promoter.

1-34. (canceled)
 35. A culture medium comprising soy hydrolysate, yeastextract, and glucose, wherein the medium comprises a recombinantClostridium difficile cell.
 36. The medium according to claim 35,further comprising polyethylene glycol
 2000. 37. The medium according toclaim 35, further comprising thiamphenicol.
 38. The medium according toclaim 35, wherein the cell is derived from C. difficile VPI
 11186. 39.The medium according to claim 35, wherein the cell comprises apolynucleotide encoding a mutant C. difficile toxin, wherein the cellfurther comprises a Clostridium sporogenes feredoxin promoter.
 40. Themedium according to claim 35, wherein the cell comprises a nucleic acidsequence that encodes an isolated polypeptide, which comprises the aminoacid sequence set forth in any of SEQ ID NOs: 1-761.
 41. A method ofproducing a Clostridium difficile toxin comprising culturing a C.difficile cell in a medium under suitable conditions to produce a toxin,and isolating the toxin from the medium; wherein the medium comprisessoy hydrolysate, yeast extract, and glucose, and wherein the mediumcomprises a Clostridium difficile cell derived from VPI 11186, whereinthe cell lacks an endogenous polynucleotide encoding a toxin, whereinthe cell further comprises a Clostridium sporogenes feredoxin promoter.42. The method according to claim 41, wherein the cell is derived from aClostridium difficile cell selected from the group consisting ofClostridium difficile 1351, Clostridium difficile 3232, Clostridiumdifficile 7322, Clostridium difficile 5036, Clostridium difficile 4811,and Clostridium difficile VPI
 11186. 43. The method according to claim42, wherein the cell is a Clostridium difficile VPI 11186 cell.
 44. Themethod according to claim 41, wherein a sporulation gene of theClostridium difficile cell is inactivated.
 45. The method according toclaim 41, wherein the cell comprises a polynucleotide which encodes atoxin comprising the amino acid sequence set forth in any of SEQ ID NOs:1-761.
 46. The method according to claim 45, wherein the toxin comprisesthe amino acid sequence set forth in SEQ ID NO:
 84. 47. The methodaccording to claim 45, wherein the toxin comprises the amino acidsequence set forth in SEQ ID NO:
 86. 48. The method according to claim41, wherein the method further comprises isolating the C. difficiletoxin and chemically modifying at least one amino acid side chain of thetoxin.
 49. The method according to claim 47, wherein the methodcomprises contacting at least one amino acid side chain of thepolypeptide with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC).50. The method according to claim 47, wherein the method comprisescontacting at least one amino acid side chain of the polypeptide withN-Hydroxysuccinimide (NHS).